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

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 architecture of the norepinephrine transporter gene SLC6A2 in four populations

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

Abstract The norepinephrine transporter (NET) regu- differed across populations. The SLC6A2 haplotype lates levels of monoamine neurotransmitters integral to map and 25-marker panel (excluding the monomorphic a variety of behaviors and autonomic functions. Two one) is a comprehensive tool for genetic linkage studies SLC6A2 polymorphisms have been used in genetic on phenotypes related to NET function. association studies, generating intriguing but nondefin- itive results on traits such as hypertension and mood. Keywords Single-nucleotide polymorphism Æ Linkage One of these SLC6A2 variants is functional but rare. disequilibrium Æ Haplotype Æ Norepinephrine The other is common but not informative over the entire transporter Æ SLC6A2 48 kb SLC6A2 region and is insufficient to capture the functional diversity potentially contained within any SLC6A2 region. To elucidate SLC6A2 haplotype structure and define markers sufficient to capture Introduction haplotype diversity within detected haplotype blocks, 26 single-nucleotide polymorphisms (SNPs) were geno- In humans, norepinephrine (NE) is essential to fun- typed in 384 individuals evenly divided across Finnish damental cognitive and emotional processes including Caucasian, US Caucasian, Plains American Indian, and attention, learning and memory, perception of African American populations. Three conserved blocks, emotions, and perception of pain (Foote et al. 1983; 13.6, 12.5, and 25 kb in size and showing little evidence Jasmin et al. 2002). NE is also involved in autonomic for historical recombination were observed in all popu- control via its actions in the brainstem and as the lations. Haplotype diversity in block 1 and numbers of primary neurotransmitter at postganglionic sympa- common were highest in African Americans, thetic nerve terminals (Hahn et al. 2003). The majority among whom 5–6 optimal markers were sufficient to of brain noradrenergic neurons are concentrated in the maximize diversity of each block. For other populations, locus coeruleus, a phylogenetically ancient and devel- 2–3 markers/block sufficed, but the optimal markers opmentally precocious structure. These NE neurons project to limbic regions critical to cognition and affect. Parts of this work were presented at the 53rd Annual Meeting of NE released at central and peripheral synapses is American Society of Human Genetics, Los Angeles, CA, USA, November 2003. inactivated through active transport into terminals by the presynaptically localized norepinephrine transporter I. Belfer Æ G. Phillips Æ H. Hipp Æ M. B. Max (NET) (Iversen 1974). NET recaptures as much as 90% Pain and Neurosensory Mechanisms Branch, of released NE making it a critical mediator of NE National Institute of Dental and Craniofacial Research, Bethesda, MD, USA inactivation and presynaptic catecholamine homeostasis (Schomig et al. 1989). Thus, NET plays a role in I. Belfer (&) Æ G. Phillips Æ J. Taubman Æ H. Hipp Æ controlling the intensity and duration of signal trans- R. H. Lipsky Æ M.-A. Enoch Æ D. Goldman Laboratory of Neurogenetics, duction (Zahniser et al. 2001). NE interacts with many Department of Health and Human Services, other neurotransmitters both in normal cortical regula- National Institute on Alcohol Abuse and Alcoholism, tion and in the therapeutic response to psychoactive National Institutes of Health, Bethesda, MD compounds, and one critical interacting neurotransmit- 20892-1258, USA E-mail: [email protected] ter is dopamine (Jordan et al. 1994). Dopamine is the Tel.: +1-301-402-8323 NE precursor so that levels of both neurotransmitters Fax: +1-301-443-8579 are regulated by common factors, for example, tyrosine 233

Table 1 Primer and probe sequences for 5¢ nuclease SNP Primers and probes Sequences genotyping of 26 SLC6A2 single-nucleotide 1 Forward primer AAGGTCATCTCATAACCTAGCAGAGA polymorphisms (SNPs) Reverse primer TCCCTAAGACGCAACTCTAGCA Allele 1 probe (FAM) CACAGAGCTCCATGTGG Allele 2 probe (VIC) CAGAGCCCCATGTGG 2 Forward primer GGACAACAAAGCACAAAGAGTGT Reverse primer GCTTGTGTGGCCAGGATGT Allele 1 probe (FAM) TCAGCTGTTTTAATGAAG Allele 2 probe (VIC) CAGCTGTTTTGATGAAG 3 Forward primer GCTTGTGTGGCCAGGATGT Reverse primer CCAGACAAGTGCCAGCTCTT Allele 1 probe (FAM) CAGAGGGAGACTCCT Allele 2 probe (VIC) AGAGGGAGGCTCCT 4 Forward primer AAGGCACTGATACACGTAGTCTCTA Reverse primer AAACCCATTCATGAACACTTAGTTCTGA Allele 1 probe (FAM) CTCCCGATCCACTGTAG Allele 2 probe (VIC) CCCGATCCGCTGTAG 5 Forward primer GATCATAGATACACCCACCTCCCTTA Reverse primer AGTTCTGAATGCTTTCTTCCATGAA Allele 1 probe (FAM) ATGCTACGAATTATG Allele 2 probe (VIC) AATGCTACAAATTATG 6 Forward primer TCCGACCTCATGAACCTAGCTT Reverse primer CTGTCCCCGCTGTTTTGG Allele 1 probe (FAM) CTCTCCCACTTTGG Allele 2 probe (VIC) TCTCTCTCACTTTGGG 7 Forward primer GGTTCAGAACACATGGCTTGAGA Reverse primer CTGCAGGGAATCAGGAAAGC Allele 1 probe (FAM) CCCACAAGTACCTCCAC Allele 2 probe (VIC) CACAAGTGCCTCCAC 8 Forward primer CCTCACCCAGCTCCATCCT Reverse primer GGAGACTGTGCTGGAAGTTCGT Allele 1 probe (FAM) CATGCAGGGCTTC Allele 2 probe (VIC) ACATGCAGGACTTC 9 Forward primer CCCAGGTGACCCAGAGC Reverse primer CCCAGGTGACCCAGAGC Allele 1 probe (FAM) TTTCCTTCTCGCCCTGTT Allele 2 probe (VIC) CTTTCCTTCTCCCCCTGTT 10 Forward primer AGTTTCCGGTGTCGCTTCAG Reverse primer CCAGATGGGAGGCATGGA Allele 1 probe (FAM) CAGGCCCGTGATGA Allele 2 probe (VIC) AGGCCTGTGATGACA 11 Forward primer TGAACACTAGGCCAGCTCCTGTA Reverse primer GCAGCTCAGACCAATGGTTTC Allele 1 probe (FAM) AAACAGCAAGGACC Allele 2 probe (VIC) AGAAACATCAAGGACC 12 Forward primer TGCTTCCTGAACTGAGCTAGATCAT Reverse primer GCCTGGCAGGGTCAGAAGT Allele 1 probe (FAM) TCTCCCCTGCAGTCT Allele 2 probe (VIC) AGTCTCCCTTGCAGTC 13 Forward primer CACTACCAAATTCACCCACTTTCAC Reverse primer CCCCAACACCCTCATCCA Allele 1 probe (FAM) CAGAGGCCCCAACT Allele 2 probe (VIC) AGGCCCAAACTAT 14 Forward primer AATCAGGCTAAGGTCAAGGTAGTCA Reverse primer CCCCCTTAGGGATATGGGAAGATT Allele 1 probe (FAM) TTCAGGTGAGTCCTC Allele 2 probe (VIC) TGTTTCAGCTGAGTCC 15 Forward primer TCACTTTAAATCCATACCTGCTGGTA Reverse primer GCCATTGGCTTGAATTTGGTA Allele 1 probe (FAM) ACCACACAAATTAT Allele 2 probe (VIC) CCACACAGATTATC 16 Forward primer CTTAGAGATTGCTGTAACTGAAACAAAAA Reverse primer AAAGTCTAGATAATAAGAGTTCATTGCACAA Allele 1 probe (FAM) TCCCAAGAAACAGCT Allele 2 probe (VIC) TCCCAAGAGACAGCT 17 Forward primer GAGGCCCTTGGCAGTATCC Reverse primer AGTTCCTCATATTTTCCTGCCAAA Allele 1 probe (FAM) CCTGGTCTCTGTTATT Allele 2 probe (VIC) TGGTCTCTGCTATTAG 234

Table 1 (Continued) SNP Primers and probes Sequences

18 Forward primer GGCTCCAGGCCAGAAAGG Reverse primer GGCCTCTGCAAGTAGATAGGAGTTC Allele 1 probe (FAM) AGGGCGGCATGAG Allele 2 probe (VIC) AGGGCAGCATGAGA 19 Forward primer GCTCTTCCCTTCTCTGACATCATC Reverse primer GCGTGGGTAGTCAGGAAAGG Allele 1 probe (FAM) AGGTGACATAGGAACT Allele 2 probe (VIC) AGGTGACGTAGGAAC 20 Forward primer CTGAAACACAAGAAGAGCGATAGATT Reverse primer GGGATTGGCTTCAGGTATAGATAGAT Allele 1 probe (FAM) CCAGACATCATTATTT Allele 2 probe (VIC) CAGACAACATTATTTG 21 Forward primer TACAGGTGGCTGAAGCAAAGG Reverse primer GGACCCCATCAAGTTTCTGTGA Allele 1 probe (FAM) CAGGGAAGTTACCC Allele 2 probe (VIC) AGGGAAGTCACCCAC 22 Forward primer CACTTTGGCCAGTTTCCTCAAC Reverse primer AACTGGGCAGTTTACCTATGTGTTG Allele 1 probe (FAM) AAACCCCTGGGTGAC Allele 2 probe (VIC) TAAACCTCTGGGTGACAC 23 Forward primer CATGACCCAAACACCTCTCACTAG Reverse primer TGTCTCCACTAAAACTCCTGTTGAAG Allele 1 probe (FAM) CACTCCCACACTGG Allele 2 probe (VIC) ACTCCCAAACTGG 24 Forward primer AGCACACTGAGGGCTGTTAAATAAC Reverse primer CCTGGCTCAGGGTTACACTGA Allele 1 probe (FAM) CTGGGTAAGTCCTGG Allele 2 probe (VIC) TGGGTGAGTCCTGG 25 Forward primer CCAAACAGCCATGCTTAAACCT Reverse primer AGCTAATCCAGAGGTGAAACTTTCC Allele 1 probe (FAM) AGTAACGCCTGAGAGG Allele 2 probe (VIC) AACGCTTGAGAGGC 26 Forward primer TCCTACCCATCCTCCAAAACC Reverse primer GGTTATCAGGGAAGGCTTCACA Allele 1 probe (FAM) CCAGTCACCCTCAC Allele 2 probe (VIC) TCCAGTCAACCTCA

hydroxylase activity. The NET has the ability to trans- (dopamine transporter) (Nelson 1998; Hahn and Blakely port dopamine, and drugs that block the NET increase 2002). SLC6A2 has five alternative splice transcripts. extracellular levels of both NE and DA (Tanda et al. Resequencing of SLC6A2identified 13 DNA sequence 1997; Bymaster et al. 2002; Gu et al. 1996). Monoamine variants, among them five low-frequency missense sub- transporters are initial sites of action for several anti- stitutions (Stober et al. 1996). The reported missense depressant drugs (including several which are relatively substitutions Val69Ile, Thr99Ile, Val245Ile, Val449Ile, NET selective) as well as psychostimulants including and Gly478Ser are located in putative transmembrane cocaine and the amphetamines (Pacholczyk et al. 1991; domains 1, 2, 4, 9, and 10, respectively. The Thr99Ile Ritz et al. 1990; Tatsumi et al. 1997; Sacchetti et al. substitution is at the 5th position of a putative leucine 1999). Decreases in NE uptake sites and activity zipper in transmembrane domain 2. A rare Ala457Pro have been observed in hypertension, diabetes, cardio- substitution in exon 9 resulting in more than 98% loss of myopathy, and heart failure (Esler et al. 1981; Merlet function has recently been detected (Ivancsits et al. et al. 1992; Bohm et al. 1995; Schnell et al. 1996; Backs 2003). A synonymous substitution also located in exon et al. 2001), and insufficient NE clearance may contrib- 9, A1287G, has been used in a series of association ute to the progression of these diseases (Bohm et al. studies to NE-related phenotypes including hyperten- 1998). sion and mood disorders (Stober et al. 1996; The human NET gene (SLC6A2, hCG2025341) is Leszczynska-Rodziewicz et al. 2002; Samochowiec et al. located on chromosome 16q12.2 (Bru¨ss et al. 1993) and 2002). However, no common functional NET poly- has 15 exons spanning 48 kb (Po¨rzgen et al. 1995, morphisms are known. 1998). The cDNA sequence encodes a 617-amino acid The NET markers used so far in linkage studies do protein with 12 highly hydrophobic membrane domains not capture the potential information on NET func- and a high level of amino acid identity to other members tional variation, and the results obtained so far have of the Na+/Cl)-dependent monoamine transporter been nondefinitive. A haplotype approach combining family, e.g., HTT(serotonin transporter) and DAT abundant missense polymorphisms with a series of loci 235 chosen for haplotype informativeness offers the poten- obtained according to human research protocols ap- tial for detection of effects of any allele of moderate proved by the human research committees of the abundance and effect size, regardless of whether the al- recruiting institutes, including the National Institute on lele is presently known or unknown (Gabriel et al. 2002). Alcohol Abuse and Alcoholism, National Institute of Concerning linkage disequilibrium (LD), many regions Mental Health, Rutgers University, and University of of the have a block-like structure such that all Helsinki. All participants had been psychiatrically loci within the block region tend to be in strong LD. interviewed, and none had been diagnosed with a psy- However, haplotype block boundaries, strength of LD, chiatric disorder. haplotype diversity, and optimal marker panels to fully capture haplotype diversity vary across populations. In this study, we report the haplotype structure of SNP markers The physical position and frequency of SLC6A2obtained using 26 single-nucleotide polymor- minor alleles (>0.05) from a commercial database phisms (SNPs) genotyped in four populations: Finnish (Celera Discovery System, CDS, July, 2003) were used and American Caucasians, American Indians, and to select SNPs (including A1287G and Ala457Pro). A African Americans. We also describe marker panels for total of 50 SLC6A2 SNPs were identified in the data- each block, which maximize haplotype information base. 5¢ nuclease assays (vide infra) could be designed for content. 35, and of these, 26 SNP assays detected sequence polymorphisms and could be genotyped in highly accurate fashion. This panel of 26 equally spaced markers covered the 48-kb gene plus 4 kb upstream and Materials and methods 4 kb downstream.

Participants A total of 384 participants were genotyped, including 96 individuals from each of four Genomic DNA Genomic DNA was extracted from populations: Finns, US Caucasians, African Americans, lymphoblastoid cell lines and diluted to a concentration and Plains American Indians. Informed consent was of 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-nucleotide polymorphism (SNP) marker with location shown in Fig. 1. Orientation is #26–#1 (5¢–3¢)

# SNP ID SNP ID Variation Position Location Allelic frequency (for allele 2) (CDS) (NCBI) (CDS) US Caucasians Finnish Plains African Caucasians Indians Americans

1 hCV1232486 rs258099 T>C 42997914 3¢ intergenic 0.67 0.71 0.68 0.17 2 hCV1232479 rs171798 A>G 43000649 3¢ intergenic 0.82 0.78 0.88 0.46 3 hCV1232477 rs7188230 A>G 43001386 3¢ intergenic 0.81 0.76 0.97 0.43 4 hCV26354929 no rs A>G 43003652 3¢ intergenic 0.16 0.27 0.02 0.34 5 hCV11291484 no rs G>A 43004999 3¢ intergenic 0.86 0.83 0.98 0.71 6 hCV1232474 rs15534 C>T 43008627 3¢ UTR 0.13 0.17 0.02 0.36 7 hCV26354922 rs8049681 A>G 43009804 Intron 14 0.13 0.16 0.02 0.38 8 hCV1232472 rs1800887 G>A 43011565 Intron 11 0.83 0.73 0.97 0.47 9 Ala457Pro no rs G>C 43013237 Exon 9 1.00 1.00 1.00 1.00 10 hCV3020068 rs5569 C>T 43013319 Exon 9 0.27 0.38 0.44 0.11 11 hCV1232470 rs5568 G>T 43015030 Intron 7 0.62 0.66 0.77 0.86 12 hCV1232469 rs1861647 C>T 43016748 Intron 6 0.31 0.40 0.45 0.12 13 hCV3020083 rs47958 C>A 43018692 Intron 6 0.47 0.42 0.43 0.31 14 hCV1232465 no rs G>C 43020661 Intron 5 0.68 0.60 0.56 0.89 15 hCV3020076 rs1345429 A>G 43022288 Intron 5 0.52 0.47 0.54 0.40 16 hCV1232458 rs40616 A>G 43023961 Intron 5 0.51 0.56 0.55 0.63 17 hCV11291524 no rs T>C 43025783 Intron 4 0.70 0.63 0.56 0.87 18 hCV3020077 rs1875544 G>A 43033100 Intron 4 0.25 0.34 0.37 0.09 19 hCV1232444 rs187714 A>G 43038757 Intron 4 0.29 0.42 0.46 0.25 20 hCV7719239 rs933555 T>A 43040802 Intron 3 0.73 0.59 0.55 0.74 21 hCV1232442 rs41154 T>C 43042550 Intron 2 0.28 0.42 0.47 0.23 22 hCV1232440 rs6499771 C>T 43044584 Intron 2 0.82 0.82 0.84 0.84 23 hCV1232433 rs36028 C>A 43048097 Intron 2 0.21 0.15 0.08 0.18 24 hCV1232429 no rs A>G 43050568 Intron 2 0.87 0.85 0.86 0.95 25 hCV1232426 rs36031 C>T 43052435 Intron 2 0.71 0.59 0.53 0.67 26 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 for Biotechnology Information database, November 2003 236

Fig. 1 Location of single-nucleotide polymorphisms (SNPs) geno- was mixed with 900 nmol of each forward and reverse typed in the human SLC6A2 gene. Coding exons are shown as solid primer and 100 nmol of each reporter and quencher blocks;5¢ and 3¢ UTRs are indicated by unfilled rectangles. Physical locations are from the Celera Discovery System [CDS] database, probe. DNA was allowed to stand at 50C for 2 min and July 2003 at 95C for 10 min, amplified by 40 cycles at 95C for 15 s and 60C for 1 min, and then held at 4C. PCR was carried out with a GeneAmp PCR system 9700 (Applied Polymerase chain reaction (PCR) amplification Geno- Biosystems). typing was performed by the 5¢ nuclease method (Shi Allele-specific signals were distinguished by measuring et al. 1999) using fluorogenic allele-specific probes. Oli- endpoint 6-FAM or VIC fluorescence intensities at 508 gonucleotide primer and probe sets were designed based and 560 nm, respectively, and genotypes were generated on gene sequence from the CDS, July 2003. Primers and using Sequence Detection System Software Version 1.7 detection probes for each locus are listed in Table 1. (Applied Biosystems). Genotyping error rate was directly In each reaction well, 2.5 ll of PCR Master Mix determined by regenotyping 25% of the samples, (Applied Biosystems, CA, USA) containing AmpliTaq randomly chosen, for each locus. The overall error rate Gold DNA Polymerase, dNTPs, gold buffer, and MgCl2 was <0.005. Genotype completion rate was 0.98.

Table 3 Pairwise linkage disequilibrium (D¢) among eight U.S. Caucasians marker 1 2 3 4 5678 single-nucleotide 1 1.00 0.66 0.66 0.65 0.60 0.60 0.66 polymorphisms (SNPs) in 2 0.71 0.71 0.70 0.66 0.66 0.71 haplotype block 1 across four 3 1.00 1.00 1.00 1.00 1.00 populations 4 0.86 1.00 1.00 1.00 5 1.00 1.00 0.86 6 1.00 1.00 7 1.00 8 Finnish Caucasians marker 1 2 3 4 5678 1 1.00 0.64 0.55 0.27 0.30 0.30 0.54 2 0.79 0.79 0.34 0.37 0.37 0.82 3 1.00 0.82 0.86 0.86 1.00 4 0.95 1.00 1.00 1.00 5 1.00 1.00 0.95 6 1.00 1.00 7 1.00 8 Plains Indians marker 1 2 3 4 5678 1 1.00 0.40 0.25 0.00 0.00 0.00 0.40 2 0.32 1.00 1.00 1.00 1.00 0.32 3 0.74 1.00 1.00 1.00 1.00 4 1.00 1.00 1.00 0.74 5 1.00 1.00 1.00 6 1.00 1.00 7 1.00 8 African Americans marker 1 2 3 4 5678 1 1.00 1.00 1.00 0.88 1.00 1.00 1.00 2 0.81 0.92 0.83 0.93 0.93 0.81 3 1.00 0.82 1.00 0.93 0.75 4 0.88 0.97 0.97 1.00 5 0.87 0.87 0.87 6 0.97 0.97 7 1.00 8 237

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

U.S. Caucasians marker 10 11 12 13 14 15 16 17 10 0.83 0.94 0.91 0.88 0.92 0.91 0.94 11 0.90 0.94 0.90 0.93 0.94 0.89 12 1.00 1.00 0.97 0.89 1.00 13 1.00 1.00 1.00 1.00 14 0.96 0.88 0.94 15 0.95 0.96 16 0.88 17 Finnish Caucasians marker 10 11 12 13 14 15 16 17 10 0.57 1.00 1.00 1.00 1.00 0.97 1.00 11 0.60 0.72 0.61 0.70 0.71 0.58 12 1.00 1.00 1.00 0.97 1.00 13 1.00 1.00 1.00 1.00 14 0.97 0.97 1.00 15 0.98 1.00 16 0.97 17 Plains Indians marker 10 11 12 13 14 15 16 17 10 0.94 0.98 0.97 0.95 0.95 0.94 0.98 11 0.94 1.00 1.00 1.00 1.00 1.00 12 0.97 0.98 0.95 0.94 1.00 13 0.97 1.00 0.98 1.00 14 0.95 0.94 1.00 15 0.97 0.98 16 0.97 17 African Americans marker 10 11 12 13 14 15 16 17 10 1.00 0.95 1.00 0.95 1.00 0.75 0.95 11 1.00 0.88 1.00 1.00 1.00 1.00 12 1.00 1.00 1.00 0.75 1.00 13 1.00 0.94 0.92 0.86 14 1.00 0.75 1.00 15 0.93 0.89 16 0.76 17

Haplotype analysis Haplotype frequencies were esti- Within SLC6A2, three conserved LD blocks (1, mated using a Bayesian approach implemented with 13.6 kb; 2, 12.5 kb; 3, 25 kb) were observed in all four PHASE (Stephens et al. 2001). These frequencies closely populations. Definition of haplotype blocks and block agreed with results from a maximum likelihood method boundaries is an inexact science. Some disruptions of D¢ implemented via an expectation–maximization (EM) (a measure of LD) occurring within blocks are clearly algorithm (Long et al. 1995). attributable to low allele frequencies that lead to in- creased variance in estimation of D¢. We discounted low D¢ values, which appeared to originate from this cause. Results and discussion In the SLC6A2 haplotype block regions, D¢ was gener- ally >0.85 from one end of the region to the other. D¢ Of a total of 26 SLC6A2SNPs, 25 were polymorphic in averaged was 0.83, 0.94 and 0.94 in blocks 1, 2, and 3, all four populations. Dramatic interpopulation differ- and perhaps more importantly, the median D¢ value ences in allele frequencies were observed for most of the within haplotype blocks was 0.97, 0.97, and 1.00 for SNPs. Ala457Pro, the previously reported in vitro blocks 1, 2, and 3, meaning that most of the SNP loci functional variant, was monomorphic in the 384 indi- were in very high LD. We note that in the situation that viduals representing the four populations. Allele fre- haplotype block boundaries are drawn too widely, an quencies of SLC6A2 SNPs and their locations in the increased number of haplotypes will be observed for the gene are shown in Table 2. The majority of the markers block and an increased number of markers will be are located in the intronic sequence of the gene, one required to capture this diversity. SLC6A2 haplotype synonymous substitution (A1287G) is located in exon 9, block boundaries could be drawn somewhat differently one marker is located in the 3¢ UTR region, five are in than we have done, and it can also be observed that the 3¢ region, and one is in the 5¢ region (Fig. 1). All there is some variation from population to population. genotype frequencies conformed to Hardy–Weinberg For example, there was some disruption of LD within equilibrium. block 1 in both Finns and Plains Indians. However, the 238

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

U.S. Caucasians marker 18 19 20 21 22 23 24 25 26 18 0.97 0.97 0.97 1.00 0.89 1.00 0.97 0.96 19 1.00 1.00 1.00 1.00 1.00 1.00 0.97 20 1.00 1.00 1.00 1.00 1.00 1.00 21 1.00 1.00 1.00 1.00 1.00 22 0.85 1.00 1.00 0.45 23 1.00 1.00 0.75 24 1.00 1.00 25 1.00 26 Finnish Caucasians marker 18 19 20 21 22 23 24 25 26 18 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 19 1.00 1.00 1.00 1.00 1.00 1.00 1.00 20 1.00 1.00 1.00 1.00 1.00 1.00 21 1.00 1.00 1.00 1.00 1.00 22 1.00 1.00 1.00 0.86 23 1.00 1.00 0.76 24 1.00 1.00 25 1.00 26 Plains Indians marker 18 19 20 21 22 23 24 25 26 18 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.94 19 1.00 1.00 1.00 1.00 1.00 1.00 0.95 20 1.00 1.00 1.00 1.00 1.00 0.95 21 1.00 1.00 1.00 1.00 0.95 22 1.00 1.00 1.00 1.00 23 1.00 1.00 1.00 24 1.00 1.00 25 0.95 26 African Americans marker 18 19 20 21 22 23 24 25 26 18 1.00 1.00 1.00 0.90 0.97 0.24 1.00 0.87 19 0.86 0.87 0.96 0.96 0.75 0.95 0.64 20 1.00 0.73 0.97 0.75 0.88 0.61 21 0.72 0.97 0.75 0.91 0.63 22 0.96 0.94 0.73 0.62 23 0.96 0.86 0.58 24 0.82 0.79 25 0.34 26

marker panels we genotyped were sufficient to capture 9 for blocks 1, 2, and 3, respectively. Knowing this, the diversity in the blocks in the four populations we stud- value of additional SNP markers for haplotype diver- ied, as described below. sity (informativeness) can then be evaluated. We began Pairwise LD values within each haplotype block are with the haplotypes derived from all available markers summarized in Tables 3, 4, and 5; all pairwise LD values in the block and successively subtracted markers, first among 25 SNPs across four populations are represented subtracting markers that resulted in no change what- in Tables 6, 7, 8, and 9. Haplotype frequencies for the ever in haplotype diversity and then subtracting three blocks in four populations are shown in Table 10. markers which changed diversity in the most minimal For each population and haplotype block, 3–6 common fashion and so on until we were left with a single, (‡0.05) haplotypes accounted for most of the total: 85– independent, highest heterozygosity SNP marker. The 96% of Caucasian and Plains Indian haplotypes and 75– figures graphically depict a reversal of this process, 89% of African American haplotypes. The number of showing that diversity can be maximized using a common (‡0.05) haplotypes were block 1: 4, 5, 3, 6; smaller group of tag SNPs selected from a larger panel. block 2: 5, 5, 4, 5, and block 3: 4, 5, 5, 6 for U.S. and After each marker addition using the pathway deter- Finnish Caucasians, Plains Indians, and African Amer- mined by the subtraction analysis, haplotype frequen- icans, respectively. cies, and diplotype heterozygosity, the chosen measure For each haplotype block, a panel of markers suf- of diversity were recalculated. At some point for each ficient to maximize genetic information content was haplotype block and each population, the addition of a available. Excluding Ala457Pro, the number of SNPs new SNP marker did not appreciably increase diversity, available for the three haplotype blocks were 8, 8, and as shown in Fig. 2. Table 6 Pairwise linkage disequilibrium (D¢) among 25 SLC6A2 single-nucleotide polymorphisms (SNPs) in U.S. Caucasians. Marker #9, Ala457Pro, was monomorphic and thus excluded

123 4 5 6 7 8 1011121314151617181920212223242526

1 1.00 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 2 0.69 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 3 1.00 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 4 0.84 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 5 1.00 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 6 1.00 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 7 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 8 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 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 26 239 240

Table 7 Pairwise linkage disequilibrium (D¢) among 25 SLC6A2 single-nucleotide polymorphisms (SNPs) in Plains Indians. Marker #9, Ala457Pro, was monomorphic and thus excluded

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1 0.99 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 2 0.32 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 3 1.00 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 4 1.00 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 5 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 6 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 7 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 8 0.70 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 Table 8 Pairwise linkage disequilibrium (D¢) among 25 SLC6A2 SNPs in Finnish Caucasians. Marker #9, Ala457Pro, was monomorphic and thus excluded

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1 1.00 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 2 0.78 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 3 1.00 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 4 0.95 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 5 1.00 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 6 1.00 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 7 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 8 0.07 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 26 241 242

Table 9 Pairwise linkage disequilibrium (D¢) among 25 SLC6A2 single-nucleotide polymorphisms (SNPs) in African Americans. Marker #9, Ala457Pro, was monomorphic and thus excluded

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1 0.90 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 2 0.76 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 3 0.95 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 4 0.83 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 5 0.82 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 6 0.96 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 7 0.97 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 8 0.49 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 26 243

Table 10 Frequencies of haplotypes constructed from 25 U.S. Caucasians Finnish Caucasians Plains Indians African Americans single-nucleotide polymorphisms (SNPs)a. Common haplotypes (block 1) (1=allele 1, 2=allele 2) 22212112 0.64 0.62 0.65 0.16 12212112 0.11 0.08 0.21 0.2 11121221 0.1 0.08 0 0.26 11212112 0.05 0 0.1 0 11122111 0 0.1 0 0 22121221 0 0.06 0 0 11112111 0 0 0 0.1 11122221 0 0 0 0.06 12112112 0 0 0 0.05 Common haplotypes (block 2) 11122212 0.35 0.29 0.2 0.12 22211121 0.27 0.31 0.43 0.08 12112122 0.14 0.12 0 0.49 12122212 0.1 0.14 0.23 0.15 12112222 0.05 0 0.08 0 21211121 0 0.07 0 0 12112212 0 0 0 0.05 Common haplotypes (block 3) 112121221 0.31 0.25 0.28 0.29 221221212 0.24 0.33 0.35 0.07 112122221 0.17 0.14 0.08 0.04 a 112111121 0.13 0.16 0.15 0.04 Haplotypes were constructed 121221212 0.04 0.08 0.08 0.11 using PHASE as described in 112111221 0 0 0 0.08 ‘‘Materials and methods’’ and 112122222 0 0 0 0.13 from 192 chromosomes repre- 112121211 0 0 0 0.07 senting each population

The required number of tag SNPs varies according to For SLC6A2, the 25-locus SNP panel defines a the haplotype diversity of the region (and population) and three-block LD structure across the entire gene the information content of the markers available. Hap- region and would be sufficient to capture the signal lotype diversity in block 1 was greatest in African Amer- of any moderately abundant SNP. For example, icans, and to maximize it, more markers were needed (5–6 A1287G is the marker most extensively used in NET markers). For the other populations, 2–3 markers sufficed linkage studies, and this SNP is located in block 2, for for block 1, but the optimal markers differed across which the SLC6A2 SNP panel includes another seven populations. Thus, each SNP had different information markers in addition to A1287G. When A1287G is ex- content in different populations. For association/linkage cluded, 99% of the information content of block 2 is still studies, different tag SNPs could be used in different target captured. populations. Alternatively, the entire panel of 25 SNPs In conclusion, the SLC6A2haplotype map and mar- could be applied to reliably capture haplotype diversity ker panel are a comprehensive tool for genetic linkage across populations. As illustrated in Fig. 2, genotyping studies on phenotypes related to noradrenergic function. larger panels of markers yields a steadily diminishing re- This map is a surrogate for moderately abundant effec- turn, but another purpose of this approach is to capture tive alleles, which may be unknown or unrecognized as more information on certain rarer haplotypes. The focus functional. of haplotype-based genetic association studies has been the detection of effects of moderately abundant loci, be- Acknowledgements We are grateful to Dr. Alec Roy and Dr. Matti cause haplotypes and functional alleles of low frequency Virkkunen for subsets of their population datasets, to Longina are not well represented in small datasets. However, Akhtar for assistance with cell culture, and Ilona Lorincz for power increases in larger datasets that may be available technical assistance. for certain noradrenergic related phenotypes, for exam- Supported by NIDCR Intramural Grant Z01, NIAAA Intra- ple, diseases such as hypertension and mood disorders, mural Grant Z01 AA000301 (National Institutes of Health, Beth- esda, MD, USA), and the Comprehensive Neuroscience Program which are readily diagnosed and which afflict very large Grant USUHS G192BR-C4 (Henry Jackson Foundation, Rock- segments of populations. ville, MD, USA). 244

Fig. 2a–c Effect of successively adding optimal SNPs on haplotype diversity in four populations. The successive addition of SNPs determined by the subtraction path, which maximally retained haplotype diversity, identifies a minimal set of tag SNPs to maximize diversity (diplotype heterozygosity) within SLC6A2 haplotype blocks (a–c) in four populations

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