Alan R. Sanders,* † Qiuhe Cao,† Jennifer Taylor,† ‡ Tamara E. Levin,* Judith A. Badner,* † Anibal Cravchik,† 1 Josep M. Comeron,§ Saitou Naruya, 1 Amado Del Rosario,* Debra A. Salvi,* Katherine A. Walczyk,* Bryan J. Mowry,‡ Douglas F. Levinson,|| Raymond R. Crowe,** Jeremy M. Silverman,†† and Pablo V. Gejman* † 2 * Schizophrenia Genetics Research Program, Department of Psychiatry, The University of Chicago, Chicago, Illinois 60637; ↑ Unit on Molecular Clinical Investigation, Clinical Neurogenetics Branch, National Institute of Mental Health, Bethesda, Maryland 20892; † Department of Psychiatry, University of Queensland, and Queensland Centre for Schizophrenia Research, Wolston Park Hospital, Brisbane, Queensland, Australia: §Department of Ecology and Evolution, The University of Chicago, Chicago, Illinois 60637; "Laboratory of Evolutionary Genetics, National Institute of Genetics, Mishima, Japan; ‖Department of Psychiatry, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; ** Mental Health Clinical Research Center and Department of Psychiatry, The University of Iowa College of Medicine, lowa City, Iowa 52242; and ↑↑Department of Psychiatry, Mount Sinai School of Medicine, New York, New York 10029 Received June 5, 2000; accepted October 12, 2000 We systematically and comprehensively investi- gated polymorphisms of the HTR1B gene as well as their linkage disequilibrium and ancestral relation- ships. We have detected the following polymorphisms in our sample via denaturing gradient gel electro- phoresis, database comparisons, and/or previously published assays: G-511T, T-261G, -182INS/DEL-181, A-161T, C129T, T371G, T655C, C705T, G861C, A1099G, G1120A, and A1180G. The results of the intermarker analyses showed strong linkage disequilibrium between the C129T and the G861C polymorphisms and revealed four common haplotypes: ancestral (via chimpanzee comparisons), 129T/861C, -161T, and -182DEL-181. The results of association tests with schizophrenia were neg- ative, although A-161T had a nominal P = 0.04 via AS- PEX/sib_tdt. The expressed missense substitutions, Phe124Cys, Phe219Leu, Ile367Val, and Glu374Lys, could potentially affect ligand binding or interaction with G All database accession numbers are registered with the dbSNP (Single-Nucleotide Polymorphism Database), curated by the Na- tional Center for Biotechnology Information (NCBI at http:// www.ncbi.nlm.nih.gov/SNP). Below are our group's recommended SNP names for these polymorphisms, followed by the NCBI Assay ID numbers. accessed     by      http://www.ncbi.nlm.nih.gov/SNP/snp_ retrieve.cgi?subsnp_id=#, with the pertinent number replacing the pound sign at the end of the URL. HTR1B_G-511T, HTR1B_A-161T, HTR1B_–182INS/DEL-181, HTR1B_C129T, HTR1B_G276A, HTR1B_T371G, HTR1B_T655C, HTR1B_C705T, HTR1B_G861C, HTR1B_A1099G, HTR1B_G1120A, NCBI Assay ID 137614; NCBI Assay ID 137615; NCBI Assay ID 137616; NCBI Assay ID 137617; NCBI Assay ID 137618; NCBI Assay ID 137619; NCBI Assay ID 137620; NCBI Assay ID 137621; NCBI Assay ID 137622; NCBI Assay ID 137623; and NCBI Assay ID 137624. ¹ Current affiliation: Medical Affairs Department, Celera Genom- ics, Rockville, MD 20850. ² To whom correspondence and reprint requests should be ad- dressed at Jules F. Knapp Research Center, The University of Chicago, 924 East 57th Street, Room R-010, Chicago, IL 60637. Telephone: (773) 834-3060. Fax: (773) 834-2970. E-mail: pgejman@uchicago.edu. proteins and thus modify drug response in carriers of these variants. On average, the human cSNPs and dif- ferences among other primates clustered in the more thermodynamically unstable regions of the mRNA, which suggests that the evolutionary survival of nucle- otide sequence variation may be influenced by the mRNA structure of this gene.       2001 Academic Press
INTRODUCTION
Serotonin (HT)³ is a neurotransmitter with a critical role in various brain functions including feeding, sleep, pain, mood, aggression, thermoregulation, locomotion, and learning and in the pharmacology of depression and psychosis. HT elicits its effects through a super- family of receptors, whose common properties are that they are integral membrane proteins, have seven hy- drophobic regions, and bind the receptors with nano- molar affinity. The HTR1 subfamily of receptors con- tains the subtypes HTR1A-F, and upon activation at low concentrations of serotonin they inhibit adenylyl cyclase (Barnes and Sharp, 1999). The intronless hu- man HTR1B gene, which is located at 6q14.3–q16.3 (GDB 132312), encodes a 390-amino-acid polypeptide (Jin et al., 1992). HTR1B mRNA is predominantly present in the caudate-putamen and cortex, but it is also detected in the hippocampus, cerebellum, and ce- ³ Abbreviations used: HT, serotonin, also known as 5-hydroxytryp- tamine; HTR, serotonin receptor; HTR1B, serotonin receptor 1B, also known as serotonin receptor 1Dβ, 5HT1Dβ, and HTR1Dβ; SNP, single-nucleotide polymorphism; UTR, untranslated region; DGGE, denaturing gradient gel electrophoresis; ASA, allele-specific amplifi- cation; RFLP, restriction fragment length polymorphism; SSCP, sin- gle-strand conformational polymorphism; TDT, transmission test for linkage disequilibrium; PCR, polymerase chain reaction; INS/DEL, insertion/deletion; DHPLC, denaturing high-performance liquid chromatography; VDA, variant detector arrays. 1 0888-7543/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
² Full trios are composed of an affected offspring and both parents, and a maximum of one full trio per family is tabulated here. b Cao et al. (1997). c Cloninger et al. (1998). d Levinson et al. (1998). rebral arteries (Bruinvels et al., 1994). The HTR1B protein has both autoreceptor (presynaptic) and het- eroreceptor (postsynaptic) function in various brain ar- eas and is probably involved with arterial contractile response mechanisms and with behavioral traits such as locomotion, feeding, and thermoregulation (Barnes and Sharp, 1999); it is thought to mediate the thera- peutic effect of the agonist sumatriptan for migraine headache (Ferrari and Saxena, 1993) and is a candi- date as a molecular target for lithium action (Massot et al., 1999). Mice that lack the HTR1B gene show a variety of interesting behavioral changes, namely, in- creased aggression (Saudou et al., 1994; Zhuang et al., 1999), increased exploratory and decreased anxiety be- havior (Zhuang et al., 1999), increased motor impulsiv- ity (Brunner and Hen, 1997), facilitated learning abil- ities (Malleret et al., 1999), increased vulnerability to cocaine reinforcement (Rocha et al., 1998), elevated alcohol consumption (Crabbe et al., 1996), attenuated locomotor response to 3,4-methylenedioxymetham- phetamine (Scearce-Levie et al., 1999), disturbances in paradoxical (rapid eye movement) sleep (Boutrel et al., 1999), and differences in sensorimotor gating such as prepulse inhibition (Dulawa et al., 1997). In humans, the HTR1B gene has been reported as associated with antisocial alcoholism (Lappalainen et al., 1998). How- ever, a definitive test of association with a human disease phenotype remains elusive. Most association studies (Ohara et al., 1996; Lappalainen et al., 1998; MaassenVanDenBrink et al., 1998; Hinney et al., 1999; Huang et al., 1999; Vincent et al., 1999) typically have examined only one HTR1B gene polymorphism and have not performed haplotype analyses or taken into account the allelic distribution in various ethnic groups. A disease locus for schizophrenia probably ex- ists in 6q13–q26 (OMIM 603175), the linkage region encompassing approximately 60 cM via the initial re- port (Cao et al., 1997) and narrowed to approximately 20 cM within 6q14–q21 with an increased sample size from pooling multiple family sets recently (Martinez et al., 1999; Levinson et al., 2000). The latter large repli- cation effort consisting of 1937 affected individuals in 734 families (824 independent affected sibling pairs (ASPs), 1003 ASPs counting all possible pairs) pro- vided strong support (empirical P = 0.0004 without inclusion of the original data set) for the 6q13–q26 linkage findings (Levinson et al., 2000). This region also contains the HTR1B gene (mapped to 6q14.3– q16.3). We have screened for variants of the HTR1B gene in the same data sets (Cao et al., 1997; Cloninger et al., 1998; Levinson et al., 1998) used for the original linkage study (Cao et al., 1997) plus an additional data set (Martinez et al., 1999). To study the genetic diversity of the HTR1B gene, we have employed denaturing gradient gel electrophoresis (DGGE) analysis, database comparisons, and previ- ously published assays of the coding sequences of the HTR1B gene. Our goals were (1) to detect and study ap- polymorphisms by multiple complementary proaches, (2) to analyze their ancestral and haplotypic relationships, and (3) to perform association tests in our available schizophrenia pedigree samples.
MATERIALS AND METHODS
We screened 94 unrelated individuals, consisting of Subjects. 62% European Americans, 20% African Americans, and 18% other groups from schizophrenia pedigree collections, for polymorphisms. Analytically, the screening of 94 subjects has a probability of 0.85 of detecting at least one variant with a frequency of 0.01 and a proba- bility of 1.00 for a variant with a frequency of 0.05, given the ability to detect any variations actually present. When a DNA variant was detected, the whole pedigree collection, i.e., 830 individuals from 193 families with 267 unrelated members including families from three data sets, was genotyped (Cao et al., 1997; Cloninger et al., 1998; Levinson et al. , 1998), as described in Table 1. Since these data sets are well described in the literature (Cao et al., 1997; Cloninger et al.,
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The Sou GENETIC DIVERSITY OF THE HUMAN HTR1B GENE 4
" A of the ATG start codon is position +1. * GC-clamp sequence is 5'-GCGGCCGCCCGTCCCGGCGCCCCCCGCCCCGCCGCCGCCC-3'. 1998; Levinson et al. , 1998), a brief summary follows. The Cao et al. (1997) families contained sibships with two ( ~ 91%) or more ( ~ 9%) siblings diagnosed by DSM-III-R (American Psychiatric Association, 1987) as having schizophrenia or schizoaffective disorder (depressive or manic types). Families were recruited opportunistically through advertisements, an advocacy group (the National Alliance for the Mentally Ill (NAMI)), and clinical collaborators, and there were no known instances of both parents of an affected individual being affected themselves. The information collected to make final best estimate diagnoses consisted of a semistructured diagnostic inter- view—the Schedule for Affective Disorders and Schizophrenia–Life- time Version (Endicott and Spitzer, 1978), systematic review of med- ical records, and a family history interview (Gershon et al., 1988). The Cloninger et al. (1998) families used in this study contained sibships with two (~78%) or more (~22%) siblings diagnosed by DSM-III-R (American Psychiatric Association, 1987) as having schizophrenia or schizoaffective disorder (depressive type only), with at least one of the siblings being diagnosed as having schizophrenia. Families were recruited by a combination of mostly systematic as- certainment through screening of patients in psychiatric hospitals and clinics and some opportunistic ascertainment from other sources such as clinician referrals and NAMI, and families were excluded if both parents were schizophrenic. The information collected to make final best estimate diagnoses consisted of a structured interview— the Diagnostic Interview for Genetic Studies (Nurnberger et al., 1994), medical records review, and a family history interview—the Family Instrument for Genetic Studies. The Levinson et al. (1998) families used in this study contained sibships with two ( ～ 68%) or more (~32%) siblings diagnosed by DSM-III-R (American Psychiat- ric Association, 1987) as having schizophrenia, schizoaffective disor- der, or nonaffective psychoses (atypical nonaffective psychosis, delu- sional disorder, and schizophreniform disorder), although we used only trios from such families in the analyses (one trio per family). Families were recruited from a combination of clinical and research settings, and families were excluded if there was suspicion or dem- onstration of bilinearity within two degrees of the proband. The information collected to make final best estimate diagnoses consisted of a modified Schedule for Affective Disorders and Schizophrenia (Endicott and Spitzer, 1978) or the Comprehensive Assessment of Symptoms and History (Andreasen et al., 1992) diagnostic inter- views, and medical record review. As can be seen from the above review, the data sets were very similar with respect to source, meth- ods used, diagnostic systems, and general exclusion and inclusion criteria. To address ethnicity differences in polymorphism detection and allele frequencies, we screened with DGGE a sample of 10 different ethnic groups of 7 to 10 unrelated individuals each and genotyped this ethnic diversity panel, described in Table 2, for any polymorphisms detected in the original, largely European American sample. This ethnic diversity panel allowed us a reasonable chance (power ranging from 0.76 to 0.87) of detecting at least one variant with a frequency of 0.05 in each of the 10 different ethnic groups of 7 to 10 unrelated individuals each. If a polymorphism was evenly distributed across all of the groups, this power was 0.80 for detecting variants with minor allele frequencies down to 0.01. DNA isolation and amplification.   A 35- μ l total volume contained 50 ng genomic DNA isolated from whole blood, 12 pmol of each primer, 1.2 U Taq Gold DNA polymerase (Perkin–Elmer), 50 mM KCl, 2.5 mM MgCl 2 , 10 mM Tris–HCl (pH 8.3), 0.01% gelatin, and 0.25 mM dNTPs. Samples were processed in a GeneAmp 9600 PCR (polymerase chain reaction) System (Perkin–Elmer). Annealing tem- perature varied from 57 to 70°C. Experiments included a sample with no DNA for control of contamination. We have employed DGGE, which is based on Variant analysis. two principles: (1) thermal DNA denaturation is remarkably sensi- tive to DNA sequence and pairing and (2) partial denaturation of a DNA duplex retards its electrophoretic mobility in polyacrylamide gels. A computer algorithm (Melt87) that simulates melting contour probabilities of a DNA duplex at increasing temperatures was em- ployed (Lerman and Silverstein, 1987). Selected PCR primers used are described in Table 3. DGGE and DNA sequencing were per- formed as previously described (Cao et al. , 1997; Gejman et al. , 1998); the latter was performed on an ABI Prism 377 DNA Sequencer (Perkin–Elmer) to characterize and confirm putative polymor- phisms. Previous variant detection attempts have employed the methods of single-strand conformational polymorphism (SSCP) anal- ysis (Nöthen et al., 1994; Lappalainen et al., 1995; Huang et al., 1999), dideoxy sequencing (Ohara et al. , 1996), and denaturing high- performance liquid chromatography (DHPLC) and variant detector
6 Nöthen et al. (1994) used SSCP to scan 1800 bp (−573 to 1227) of 92 chromosomes in Germans. ʿ Lappalainen et al. (1995) used SSCP to scan 1286 bp (−40 to 1246) of 136 chromosomes (c) in Finnish (c = 84), American Indians (c = 20), and European Americans ( c = 32). d Ohara et al. (1996) used sequencing to scan 1171 bp (−4 to 1167) of 80 chromosomes in Japanese. * Cargill et al  (1999) used DHPLC and hybridization to VDAs to scan 1258 bp (1173 coding and 85 noncoding bp) of an average of 114 chromosomes in Europeans, African Americans, African Pygmies, and Asians. ' Huang et al. (1999) used SSCP to scan 1231 bp (22 to 1252) of 356 chromosomes ( c ) in European Americans ( c = 212), African Americans (c = 86), and American "other" (c = 58), and they noted C129T and G861C to be in complete linkage disequilibrium. s We used DGGE to scan 1746 bp (−566 to 1180) of 1658 chromosomes in European Americans and African Americans. arrays (VDA) (Cargill et al., 1999) to scan the coding sequence of the HTR1B gene in approximately 778 chromosomes of mostly European descent (see Table 4) and have had variable success. To find putative single-nucleotide poly- Database comparisons. morphisms (SNPs), we utilized database comparisons within the coding region and nearby noncoding region of the HTR1B gene to generate more markers for extended haplotype analyses and linkage disequilibrium approaches. We obtained nine sequences containing complete coding sequence from GenBank (M81590, D10995, L09732, M75128, M83180, M89478, M84986, AB041370, and NT_001959) and performed alignment comparisons by means of the MacVector version 6.0.1 program (Oxford Molecular) to investigate putative new polymorphisms. Database sequence was also used to infer the ancestral human allele for the identified polymorphic sites, specifically, the chim- panzee ( Pan troglodytes ) and the gorilla ( Gorilla gorilla ) HTR1B gene sequences, GenBank Accession Nos. AB041371 and AB041372, respec- tively, from the Silver Project (http://sayer.lab.nig.ac.jp/~silver/). Genotyping methods.  Several polymorphisms were assayed by standard restriction fragment length polymorphism (RFLP) (Kan and Dozy, 1978) analysis of PCR-amplified DNA segments without (G-511T, A-161T, C705T, and G1120A) or with (T371G) mutagenic primers (Vallette et al. , 1989), using digestion conditions according to the manufacturer's recommendations (New England BioLabs). Al- lele-specific assays (ASA) (Glisic and Alavantic, 1996) were used for several other polymorphisms. The insertion/deletion polymorphism was assayed by detection of fluorescently labeled PCR products on an ABI Prism 377 DNA Sequencer (Perkin–Elmer). All polymorphisms and their genotyping assays are registered with the dbSNP (Single- Nucleotide Polymorphism Database), curated by the National Center for Biotechnology Information ((NCBI) http://www.ncbi.nlm.nih.gov/ SNP), under the following identifications (all prefixed with "HTR1B_"): G-511T; –182INS/DEL-181; A-161T; C129T; G276A; T371G; T655C; C705T; G861C; A1099G; and G1120A. Statistical analysis.   For the estimation of the allele frequencies disequilibrium between polymorphisms, we used the EH program (Xie and Ott, 1993; Terwilliger and Ott, 1994). For individuals with a missing genotype in the schizophrenia families (24%), we assigned the major allele at that site to estimate the full haplotype. Genotyp- ing was 100% complete on the ethnic diversity panel. We have tested association with disease by the transmission test for linkage disequi- librium (TDT) (Spielman et al., 1993). TDT considers parents who are heterozygous for a locus and evaluates the frequency with which an allele or its alternate is transmitted to affected offspring. Non- transmitted alleles provide an internal control because they are of the same ancestry as the "case" chromosomes. TDT will detect link- age between the marker locus and the disease locus only if associa- tion (due to linkage disequilibrium) is also present. To control for the effects of known linkage in this region in the families tested, we conditioned on the observed identity by descent (IBD) sharing via the ASPEX program (Hinds and Risch, 1998), sib_tdt option, as reported in Table 5. To determine the haplotype(s) contributing the most to any increased number of transmissions of an allele to affected off- spring, we utilized the 4-locus TDT (for the most common polymor- phisms) as estimated by GENEHUNTER (Kruglyak et al., 1996; Kruglyak and Lander, 1998), which was also used to generate hap- lotypes from the linkage files. Fisher's Exact Test (Fisher, 1935) was utilized to estimate the probability of linkage disequilibrium among markers that had at least 10 individuals possessing the minor allele. In addition, Hardy–Weinberg disequilibrium was assessed by means of an exact test using a Markov chain. RNA folding analysis.  The coding region of 1173 bp for the hu- man HTR1B mRNA was analyzed by mfold version 3.0 (Mathews et al., 1999; Zuker et al., 1999). The HTR1B mRNA appears to be ~4.5 kb via Northern analysis (Hamel et al., 1993), which exceeds the capacity of the mfold server, currently 3000 bp. We selected the most stable folding (lowest final dG value) and examined the position of the polymorphic sites for pairing predictions. We also performed the same analyses as described above for 3000 bp (the limit of the mfold server) of the HTR1B mRNA sequence, which consisted of part of the in each marker, the haplotype frequency, and the testing of linkage
ª T is the number of transmissions to affected offspring from heterozygous parents for the more commonly transmitted allele. b NT is the number of nontransmissions to affected offspring from heterozygous parents for the more commonly transmitted allele. c P level refers to the nominal probability of the observation being due to chance, not conditioned by observed IBD sharing. d P level refers to the nominal probability of the observation being due to chance, conditional on the observed IBD sharing, i.e., controlling for the effects of linkage. This was performed by means of the ASPEX program, sib_tdt option. 5'-untranslated region (5'-UTR), the entire coding region, and the entire 3′-UTR, except for the poly(A) tail.
DGGE
RESULTS
Multiple DNA segments of the HTR1B gene revealed DGGE patterns consistent with DNA variations in a heteroduplex form. See Tables 3 and 4 for a summary of the DGGE fragments studied and the polymor- phisms detected. Subsequent sequence analyses dem- onstrated the presence of three new missense substi- tutions in the HTR1B gene (Table 6), located in transmembrane domain 5 (TMD5) Phe219Leu, TMD7 (Ile367Val), and in the cytoplasmic part of the receptor beyond TMD7 (Glu374Lys). Additionally, we detected another missense substitution, Phe124Cys (Nöthen et al., 1994). The newly detected synonymous variant was (Ala235Ala), and we also C705T C129T found (Ser43Ser) al., (Cargill et 1999) and G861C (Val287Val) (Lappalainen et al., 1995). We found no evidence of G276A (Ala92Ala) (Ohara et al., 1996), found thus far in Japanese populations only, in our sample. In the 5' noncoding region, we detected G-511T and T-261G (Nöthen et al., 1994). We have detected the new A-161T, which had interfered with attempts at our assessment of – 179INS/DEL-178 through the annealing step of the PCR using the orig- inally reported primers (Nöthen et al., 1994). We found
² A of the ATG start codon is position +1. The coding sequence is intronless and 1173 bp long. 6 Transitions are purines (A or C) for purines or pyrimidines (C or T) for pyrimidines; transversions are the other kind of change. c Position notes potential localization in protein for nonsynonymous changes. 6 Minor allele frequency refers to our sample of families. We detected neither − 179DEL/INS-178 nor G276A in our families. NT, not tested. ² First descriptions: A, Nöthen et al. (1994); B, Cargill et al. (1999); C, Ohara et al. (1996); D, Lappalainen et al. (1995). 6
² A of the ATG start codon is position +1. The coding sequence is intronless and 1173 bp long. 6 H. sapiens sequence from NM_000863 (human consensus sequence verified in this investigation). c P. troglodytes sequence from AB041371 (one sequence). d G. gorilla sequence from AB041372 (one sequence). no evidence of − 179INS/DEL-178 (Nöthen et al., 1994) in our samples with an improved set of PCR primers, however, we did detect the presence of − 182INS/DEL- 181 as a 2-bp insertion/deletion with approximately the same minor allele frequency. This probably is the same polymorphism as described by Nöthen et al. (1994), but with a difference in the nucleotide sequence determination. We sequenced one heterozygous member of each family segregating this size polymorphism and found that all of them were accounted for by −182INS/ DEL-181 and none by the previously reported −179INS/ DEL-178 (Nöthen et al., 1994). In the 3' noncoding re- gion, we found A1180G (Cargill et al., 1999).
Database Analysis
Via our sequence comparisons of nine sequences (seven of which were deposited in 1992) and containing complete coding sequence (M81590, D10995, L09732, M75128, M83180, M89478, M84986, AB041370, and NT_001959), we detected four SNPs. Three were cod- ing region polymorphisms: C129T (Ser43Ser) (Cargill et al., 1999), G276A (Ala92Ala) (Ohara et al., 1996), and G861C (Val287Val) (Lappalainen et al., 1995), and the other was the 3' noncoding region A1180G (Cargill et al., 1999). In addition, the possible presence of sev- eral other SNPs and/or insertion/deletions was indi- cated in the 3' and 5' noncoding regions by the same means. We therefore investigated whether any of these additional putative 3' or 5' noncoding region polymor- phisms were present in our data set by means of ASA, RFLP analysis of PCR-amplified fragments, and size determination upon the screening of 94 unrelated in- dividuals. None of the database sequence differences in the coding region, each with a minor allele found in one of the nine available database sequences, represented false positives. Uncommon (≤1% minor allele) SNPs, detected by DGGE, were not seen in the database se- quences, as expected. In the 3' noncoding region, the true positive A1180G was found in two of eight avail- able database sequences. There were three false posi- tives, represented by one (two cases) or two (one case) sequences. For database sequence differences in the 57 noncoding region, there was one true   positive, –182INS/DEL-181, and six false positives in two to four available sequences, with a decreasing amount of available database sequence as the distance from the coding region increased. Hence, 100% (3/3) of the cDNA database differences were true positives, and 18% (2/ 11) of the database differences outside of the cDNA were true positives. We have confirmed the consensus HTR1B gene sequence (NM_000863, derived from D10995) and the Sanger Centre sequence for this area (NT_001959) as containing the major allele at each polymorphic site we examined. In addition, the ances- tral human allele for each of the identified polymorphic sites appears to be that found in the consensus HTR1B gene sequence (NM_000863) from comparisons to the chimpanzee (AB041371) and gorilla (AB041372) se- quences, which both have 99.7% (1169/1173) identity with the human cDNA sequence. The nonhuman pri- mate sequence does not include enough of the 5' non- sequence to determine this for G-511T, coding –182INS/DEL-181, and A-161T sites. Also of note is the finding that, of the seven differences (7/1173 = 0.6%) between the human and the nonhuman pri- mates, six are synonymous and only one (Ser45Ala) is a missense change; see Table 7.
Association Tests among Markers
Fisher's Exact Test revealed several intermarker linkage disequilibria in the schizophrenia families; most notable was that between C129T and G861C ( DT = 0.922; Fisher's P < 0.001, still significant after Bon- ferroni correction). A-161T was in linkage disequilib- rium with both C129T ( D ′ = −0.283; Fisher's P = 0.009) and G861C ( D ′ = −0.232; Fisher's P = 0.034), and T371G was in linkage disequilibrium with G861C ( D ′ = 0.668; Fisher's P = 0.004, still significant after Bonferroni correction). Although a much reduced sam- ple size in each group resulted in decreased power to detect several intermarker linkage disequilibria, simi- lar findings were present in the ethnic diversity groups: P < 0.05 linkage disequilibrium between C129T and G861C (Atayal, Indo-Pakistani, Japanese, Karitiana, and Southern Chinese), A-161T and C129T (Karitiana), and A-161T and G861C (Karitiana). The only marker found to be in Hardy–Weinberg disequi- librium was A-161T ( P = 0.0024), which was found to originate from the European American schizophrenia families that had an excess of heterozygotes ( P = 0.002), whereas the African American schizophrenia families appeared to be in Hardy–Weinberg equilib- rium.
Additional Ethnic Groups
We found no evidence for additional polymorphisms in the coding region, besides those already discovered in the original, largely European American sample. We found –182INS/DEL-181, A-161T, C129T, and G861C in our sample of 10 different ethnic groups of 7 to 10 unrelated individuals each; see Table 2. As previously noted (Huang et al. , 1999), C129T and G861C appeared to be in total linkage disequilibrium in these ethnic groups, i.e., only two haplotypes appeared to exist: C129/G861 and 129T/861C. The frequency of the 129T/ 861C haplotype varied widely, from 5% in the Sub- Saharan African (Mbutu and Biaka) group to 81% in the North American (Pima and Jemez) group. The – 182INS/DEL-181 polymorphism was detected in only three groups: Caucasoid Indo-Pakistani, Caucasoid North of Sahara African (Egyptian, Moroccan, and Al- gerian), and Sub-Saharan African (Mbutu and Biaka) groups at 13, 7, and 10% minor allele frequency, re- spectively. Each time DEL-181 was detected (five times), it was always on the C129/G861 haplotype, as evidenced by being seen only in C129/G861 homozy- gotes. A-161T was not detected in the Southeast Asian Chinese (Chinese and South Chinese) or the Southeast Asian Taiwanese (Atayal) groups, but was found in other groups at a minor allele frequency ranging from 6 to 31%. Each time -161T was found (20 times), it was in either a C129/G861 homozygote (11 times) or a het- erozygote (C129/G861 129T/861C) (9 times); -161T was never found in the 129T/861C homozygotes. This is consistent with the possibility of T-161 existing only on the C129/G861 haplotype, however, with only unre- lated individuals in our ethnic diversity sample, this was not demonstrated definitively. Similarly, each time DEL-181 was detected (5 times), it was in either an A-161 homozygote (4 times) or an A-161T heterozy- gote (1 time); DEL-181 was never found in the -161T homozygotes. This is consistent with the possibility of DEL-181 existing only on haplotypes containing -161T; however, again with only unrelated individuals in our ethnic diversity sample, this was not demonstrated definitively. None of the rare (≤1%) polymorphisms found in the larger, predominantly European Ameri- can sample was detected in this smaller ethnic diver- sity sample, as expected (see Tables 2 and 8), although we had reasonable (>80%) power to detect a ≥1% minor allele frequency polymorphism, assuming it was evenly distributed among the groups. In the collection of schizophrenia families, the rare (≤1%) variants were found in either exclusively European Americans (-511T in one family, 371G in eight of eight families, 1099G in one family, and 1120A in one family) or exclusively African Americans (655C in one family and 705T in one family).
Association Tests for Schizophrenia
The results of association tests with schizophrenia in the linked data sets were negative. No polymorphism was detected primarily in affected individuals, sug- gesting that these polymorphisms do not have a large effect on disease. We analyzed the transmission of the biallelic polymorphisms located in the HTR1B gene, for which A-161T, C129T, and G861C had by far the most informative parent–offspring combinations. The ob- served number of transmissions to affected offspring was not significantly different from what would be expected by chance after correcting for multiple test- ing, although A-161T had 108 transmissions of the A-161 allele to affected offspring versus 78 nontrans- missions (nominal P = 0.028 unconditioned by ob- served IBD sharing and P = 0.041 controlling for the effects of linkage via ASPEX/sib_tdt) (Table 5). Since most polymorphisms were rare (≤1%), we also exam- ined for association of any variant allele with schizo- phrenia, which interestingly revealed 222 transmis- sions to affected offspring versus 170 nontransmissions (nominal P = 0.0086 unconditioned by observed IBD sharing and P = 0.044 controlling for the effects of link- age via ASPEX/sib_tdt) (Table 5). Via GENEHUNTER 4 locus TDT (−182INS/DEL-181_A-161T_C129T_G861C), we determined the haplotypes contributing the most to the larger number of transmissions of the A-161 allele to affected offspring: there were 19 transmissions of −182INS-181_A-161_129T_861C versus 5 nontransmis- sions (nominal P = 0.0043), while there were 2 trans- missions of − 182INS-181_-161T_129T_861C versus 8 nontransmissions (nominal P = 0.058). Hence, the possible transmission distortion of the A-161T polymorphism appears to act primarily upon the −182INS-181_ 129T_861C haplotype.
RNA Folding Analysis
Mfold analysis revealed that in the resultant struc- ture of the coding region mRNA of the HTR1B gene, the polymorphic sites appeared to be preferentially located in the periphery of the molecule (see Fig. 1). This was also the case for the sites that differ between the human and the chimpanzee or gorilla HTR1B gene coding sequence (see Fig. 1). Mfold returned 41 com- peting RNA secondary structures for the coding region mRNA of the HTR1B gene, ranging in final dG from −373.94 (Fig. 1) to −353.16 (least stable). The pattern described above held for the majority of these foldings, especially the more stable foldings (data not shown). Similar patterns were found when the entire 3000-bp capacity of the mfold server was utilized in the exam-
² A of the ATG start codon is position + 1. ³ SZ (schizophrenia or schizoaffective disorder) marry-in parents have no known family history of the illness themselves. ination of 3000-bp comprising part of the 5'-UTR, the entire coding region, and the entire 3'-UTR of the HTR1B mRNA (data not shown).
DISCUSSION
Nucleotide Diversity
We have performed an intensive DNA variation analysis by DGGE of genomic sequences of the HTR1B gene and have detected 12 polymorphisms, 10 of which we genotyped. The cSNPs had a mean minor allele frequency of 8%, as opposed to those detected in non- coding regions (12%). Also of note is that 6 of the 8 currently known polymorphic sites in the coding region represent transitions, rather than transversions (see Table 6), consistent with known patterns of mamma- lian genome evolution (Epstein et al., 2000). The ratios of transitions to transversions in the cDNA (6:2) versus the noncoding regions (1:3) of this gene were not sta- tistically different (Fisher's Exact Test, P = 0.14), quite possibly due to the relatively small number of observa- tions. We found an average of one SNP per 159 bp, on par with previous reports of larger surveys a (Halushka et al., 1999), and calculated the nucleotide diversity (per basepair) in the scanned sequence of the human HTR1B gene as 7.1 × 10~ c , with the cDNAvalue being 6.8 × 10 −4 and the noncoding region being 7.8 × 10 -4. These values are consistent with previously reported observations from larger surveys (Halushka et al., 1999; Cargill et al., 1999). The actual diversity in the noncoding regions surrounding the HTR1B cDNA is likely to be higher than this value, as we did not genotype two noncoding region SNPs, T-261G in the 5'-UTR and A1180G in the 3'-UTR. Previous estimates of SNP frequency (for SNPs with ≤1% minor allele frequency) have been one per 721 bp and slightly less common than this in coding regions, i.e., for cSNPs (Wang et al., 1998). We have found six such SNPs in 1746 bp scanned for a ≤1% minor allele SNP frequency of one per 291 bp, and for such cSNPs we have found three in 1173 bp scanned for a cSNP frequency of one per 391 bp. The large sample size screened for variations allowed a large number of po- tentially functional changes to be identified. Further- more, it is improbable that a great deal more diversity in the coding region of this gene exists. These polymor- phisms and their haplotypes can be used to reach ro- bust conclusions regarding the relationship between several human disease phenotypes and the HTR1B gene. residues at positions 122 and 199, due to its close proximity to position 122.
Missense Polymorphisms
The missense SNPs were population dependent, found exclusively in either European American fami- lies (T371G, A1099G, and G1120A) or African Ameri- can families (T655C), and rarer (≤1% minor allele fre- quency), as previously described (Halushka et al., 1999). DGGE experiments detected three new mis- sense substitutions, one located in TMD5, T655C (Phe219Leu); another in TMD7, A1099G (Ile367Val); and one in the cytoplasmic part of the receptor beyond TMD7, G1120A (Glu374Lys); in addition to T371G (Phe124Cys) located in TMD3 (Nöthen et al., 1994) (refer to Fig. 2 for the illustration of these positions in the receptor). We discovered one new synonymous vari- ant, C705T (Ala235Ala), and also detected C129T (Ser43Ser) (Cargill et al., 1999) and G861C (Val287Val) (Lappalainen et al., 1995). Two of the mis- sense changes are chemically nonconservative: Phe124Cys (aromatic hydrophobic to a sulfur hydro- philic neutral residue) and Glu374Lys (basic to acidic), and another conservative: two are chemically Phe219Leu (both hydrophobic and neutral) and Ile367Val (both hydrophobic). It should also be noted that Phe124Cys potentially interferes with the normal formation of a disulfide bridge between the cysteine
Evolutionary Context
Phe124, Ile367, and Glu374 in the human are largely conserved when compared to other available mammal sequences (opossum U04311, mouse M85151, rat M89954, hamster X86458, mole rat AF027184, guinea pig U82175, rabbit U60826, gorilla AB041372, and chimpanzee AB041371) at the analogous amino acid position, with the exceptions of Leu124 in the rabbit and Asp374 in the opossum. However, this is not the case with Phe219 at which residue all primates and the opossum have Phe219 and all rodents and the rabbit have Leu219, which is the variant human amino acid at this position. Among other human HT receptors, the analogous residue to HTR1B-Ile367 varies among other hydrophobic and neutral residues, e.g., isoleucine ( HTR1B, 1D, and 6 ), valine ( HTR1A ), leucine ( HTR1E, 1F, 2A, 2B, 2C, and 5A ), and phenylalanine ( HTR7 ), providing a further suggestion that the Ile367Val change may be tolerated. The residue analogous to HTR1B-Glu374 varies among other hydrophilic resi- dues, sometimes basic and other times acidic, e.g., glu- tamic acid ( HTR1B, 1D, 1E, and 1F ), arginine ( HTR6 and 7), and lysine ( HTR1A, 2A, 2B, 2C, and 5A ), sug- gesting that acidic to basic changes here may be toler- ated. In contrast, the residue analogous to HTR1B- Phe124 varies only among other hydrophobic and neutral residues, e.g., phenylalanine ( HTR1B ), leucine ( HTR1A, 5A, and 6 ), isoleucine ( HTR1D and 1F ), va- line ( HTR1E, 2A, 2C, and 7), and alanine ( HTR2B ), suggesting that the Phe124Cys might affect the func- tion of the receptor. Finally, the residue analogous to HTR1B-Phe219 varies among other hydrophobic and neutral residues, e.g., phenylalanine ( HTR1B ), leucine ( HTR5A, and 6 ), and isoleucine ( HTR1D, 1E, 1F, 2A, 2C, and 7) except for the hydrophilic and neutral thre- onine ( HTR2B ), suggesting that the change to leucine, a neutral hydrophobic residue, is likely a functionally tolerable change. Location of Polymorphisms in the Receptor It is known that amino acid changes that modify the tertiary structure of the receptors may result in func- tional variations. Some amino acid changes in TMD6 and TMD7 are particularly important for the specific- ity of the HTR1B protein. The human HTR1B protein binds β-adrenergic antagonists with much less affinity than the rodent versions (Adham et al., 1994). This prop- erty is conferred by the amino acid residues of TMD7, and residue 355 appears to be the key position via site-di- rected mutagenesis experiments (Thr355Asn) in which the human version has been shown to become identical to the rodent version in this regard (Oksenberg et al., 1992). None of the site-directed mutagenesis experiments yet reported with the human HTR1B gene (Oksenberg et al., 1992; Granas et al., 1998a, b; Granas and Larhammar, 1999; Pauwels et al. , 1999) has targeted the residues with missense polymorphisms (Phe124Cys, Phe219Leu, Ile367Val, and Glu374Lys) for which we have detected heterozygous individuals. This would be an area meriting further exploration as has been achieved for missense variants in other genes, e.g., the human dopamine D2 receptor and dopamine D5 receptor genes (Cravchik et al., 1996, 1999; Cravchik and Gejman, 1999). Another re- lated avenue of investigation has arisen for this receptor with the development of homozygous transgenic mice that express only the human, and not the murine, HTR1B gene (Bonaventure et al., 1999).
DGGE Utility
Among the polymorphisms noted in our survey, many were not previously reported by other groups. This is not unexpected with those having uncommon minor alleles (T655C, C705T, A1099G, and G1120A) given our comparatively large sample size (see Table 4), but was somewhat surprising for the more common A-161T and −182INS/DEL-181. No previous single study has found more than four different variants, but more than one group has noted the presence of the more common polymorphisms (T-261G, C129T, T371G, G861C, and A1180G) (Table 4). The results of the present study support the usefulness of DGGE screen- ing of genes previously studied by other variant anal- ysis methods, and we report previously undetected missense, synonymous, and 5' noncoding region poly- morphisms in the HTR1B gene.
Database Comparisons
The utility of database comparisons was highest in (e.g., C129T, G276A, and G861C) or immediately adja- cent to (e.g., A1180G) the possibly higher quality se- quence of the coding region for common (e.g., C129T, G861C, and A1180G) SNPs, found more than once (e.g., A1180G). In summary, five polymorphisms (one 5' noncoding region, three coding regions, and one 3' noncoding region) were confirmed out of 13 candidates (six 5' noncoding region, three coding region, and four 3' noncoding region) for a 38% confirmation rate. This is less than some previous database screens (Picoult- Newberg et al., 1999) for SNPs that have achieved >60% confirmation rate after multiple filtering proce- dures, which we have not performed.
Linkage Disequilibrium among Markers
We have confirmed the observation of almost com- plete linkage disequilibrium between C129T and G861C (Huang et al., 1999), found evidence for linkage disequilibrium between T371G and G861C, and have also observed linkage disequilibrium to a lesser extent (no longer significant after Bonferroni corrections) be- tween A-161T and both C129T and G861C. The finding in our European American schizophrenic families of Hardy–Weinberg disequilibrium suggests further cau- tion in interpreting the nominally significant associa- tion with schizophrenia at that site (A-161T). Namely, one or more of the assumptions of Hardy–Weinberg equilibrium seem to have been violated: no natural selection, no immigration, no emigration, no mutation, random mating, and/or a large population.
Comparisons among Ethnic Groups
The C129T and G861C polymorphisms appear to be in total linkage disequilibrium with each other in mul- tiple ethnic groups, but at widely varying “minor” al- lele frequencies. −182DEL/INS-181 was detected only in Caucasoid and African groups, such as comprise most of our larger schizophrenia sample. Hence, we would have likely missed this polymorphism if we re- lied on other ethnic groups for variation scanning, and, of course, if a nearby 2-bp polymorphism had not been previously reported (Nöthen et al., 1994) to draw our attention to this area. A-161T was found in 8 of the 10 groups and would have been detectable in DGGE vari- ant scanning efforts from many various ethnic groups. We attempted to construct a cladogram for our data by parsimony and bootstrap methods, but were unable to obtain significant support for one tree versus another, except that, unsurprisingly, the chimpanzee and go- rilla sequences were always positioned as outgroups versus all of the human haplotypes (data not shown).
Linkage Disequilibrium with Schizophrenia
We have not found evidence of association of HTR1B gene polymorphisms with schizophrenia that would remain statistically significant after correcting for the multiple tests performed. However, it is quite possible that we do not have the power to detect linkage dis- equilibrium because the informative sample size is rel- atively small. Furthermore, we cannot rule out the possibility that DNA variations in regulatory se- quences (not yet examined by us or by others), not necessarily in linkage disequilibrium with the known polymorphisms, confer susceptibility to schizophrenia in a subset of patients. Nevertheless, our data and data of the previous studies tend to exclude common (and large effect) variations in the HTR1B gene in schizo- phrenia, with the possible exception of excess trans- mission of the A-161 allele to affected offspring, acting upon the −182INS-181_ 129T_861C haplotype.
RNA Folding Analysis
HTR1B gene polymorphic sites appeared to be clus- tered in the periphery of the folded mRNA molecule, as did those sites that differed between human and chim- panzee or gorilla HTR1B coding sequence (see Fig. 1). This appeared to be the case both for the coding region only and for the more complete mRNA (part of the 5'-UTR, the entire coding region, and the entire 3'- UTR, except for the poly(A) tail), up to the limits of the mfold program (Mathews et al., 1999; Zuker et al., 1999). These observations are intriguing, as they sug- gest the possibility of another route by which synony- mous variants might, at the mRNA level (e.g., through altered stability of the mRNA, or key portions thereof), lead to differences in expression of the protein. This hypothesis remains to be tested.
ACKNOWLEDGMENTS
We thank the families who participated in these studies and the many individuals who helped to advance this work, especially Elliot S. Gershon, M.D. This research was partially supported by both the Division of Intramural Research Programs of the National Institute of Mental Health and the Department of Psychiatry of the University of Chicago.