A Genomewide Scan Of Male Sexual Orientation
Hum Genet (2005) 116: 272–278DOI 10.1007/s00439-004-1241-4O RI GI N AL IN V ES T IG A T IO NBrian S. Mustanski Æ Michael G. DuPreeCaroline M. Nievergelt Æ Sven BocklandtNicholas J. Schork Æ Dean H. HamerA genomewide scan of male sexual orientationReceived: 16 September 2004 / Accepted: 30 November 2004 / Published online: 12 January 2005Ó Springer-Verlag 2005Abstract This is the first report of a full genome scan ofsexual orientation in men. A sample of 456 individualsfrom 146 families with two or more gay brothers wasgenotyped with 403 microsatellite markers at 10-cMintervals. Given that previously reported evidence ofmaternal loading of transmission of sexual orientationcould indicate epigenetic factors acting on autosomalgenes, maximum likelihood estimations (mlod) scoreswere calculated separated for maternal, paternal, andcombined transmission. The highest mlod score was3.45 at a position near D7S798 in 7q36 with approximately equivalent maternal and paternal contributions.The second highest mlod score of 1.96 was located nearD8S505 in 8p12, again with equal maternal and paternalcontributions. A maternal origin effect was found nearmarker D10S217 in 10q26, with a mlod score of 1.81 formaternal meioses and no paternal contribution. We didnot find linkage to Xq28 in the full sample, but given thepreviously reported evidence of linkage in this region, weconducted supplemental analyses to clarify these findings. First, we re-analyzed our previously reported dataand found a mlod of 6.47. We then re-analyzed ourcurrent data, after limiting the sample to those familiespreviously reported, and found a mlod of 1.99. TheseXq28 findings are discussed in detail. The results of thisfirst genome screen for normal variation in the behavioral trait of sexual orientation in males shouldencourage efforts to replicate these findings in newsamples with denser linkage maps in the suggested regions.Brian S. Mustanski and Michael G. DuPree contributed equally tothis work.IntroductionB. S. Mustanski Æ M. G. DuPree Æ S. BocklandtD. H. HamerLaboratory of Biochemistry, National Cancer Institute,National Institutes of Health, Bethesda, Md., USAAlthough most males report primarily heterosexualattractions, a significant minority (approximately 2%–6%) of males report predominantly homosexual attractions (Diamond 1993; Laumann et al. 1994; Wellingset al. 1994). Multiple lines of evidence suggest thatbiological factors play a role in explaining individualdifferences in male sexual orientation (MIM 306995).For example, the third interstitial nuclei of the humananterior hypothalamus (INAH3), which is significantlysmaller in females, is also reported to be smaller inhomosexual males (LeVay 1991). Byne and colleagues(2001) followed up on this finding by reporting a trendfor INAH3 to occupy a smaller volume in homosexualmen than in heterosexual men, with no significant difference in the number of neurons within the nucleus.Neuropsychological studies have reported differences inperformance with respect to tasks that show sex differences, such as spatial processing (e.g., Rahman andWilson 2003), which may indicate differences in relevantneural correlates (e.g., parietal cortex). The strong linkbetween adult sexual orientation and childhood gender-B. S. Mustanski (&)Institute for Juvenile Research Department of Psychiatry,University of Illinois at Chicago (M/C 747),1747 W. Roosevelt Road,Chicago, IL 60608, USAE-mail: bmustanski@psych.uic.eduTel.: 1-312-9969505M. G. DuPreeDepartment of Anthropology,Pennsylvania State University,University Park, Pa., USAC. M. Nievergelt Æ N. J. SchorkDepartment of Psychiatry,University of California,San Diego, Calif., USAS. BocklandtDepartment of Human Genetics,David Geffen School of Medicine at UCLA,Los Angeles, Calif., USA
273related traits expressed at an early age (Bailey andZucker 1995) suggests that such biological influences actearly in development, possibly prenatally. Similarly, thecorrelation between sexual orientation and a variety ofprenatally canalized anthropometric traits suggests thatsexual orientation differentiation probably occurs beforebirth (for a review, see Mustanski et al. 2002). Despitethis evidence, specific neurodevelopmental pathwayshave yet to be elucidated.Family and twin studies have provided evidence for agenetic component to male sexual orientation. Familystudies, using a variety of ascertainment strategies,document an elevation in the rate of homosexualityamong relatives of homosexual probands (for a review,see Bailey and Pillard 1995). Several family studies report evidence of increased maternal transmission ofmale homosexuality (Hamer et al. 1993; Rice et al.1999a), whereas others find no increase relative topaternal transmission (Bailey et al. 1999; McKnight andMalcolm 2000). Twin studies consistently show thatmale sexual orientation is moderately heritable (for areview, see Mustanski et al. 2002). For example, tworecent twin studies in population-based samples bothreport moderate heritability estimates, with the remaining variance being explained by nonshared environmental influences (Kendler et al. 2000; Kirk et al. 2000).The results from family and twin studies demonstratethat sexual orientation is a complex (i.e., does not showsimple Medelian inheritance) and multifactorial phenotype.A more limited number of studies have attempted tomap specific genes contributing to variation in sexualorientation. Given the evidence for increased maternaltransmission, initial efforts focused on the X chromosome. One study produced evidence of significant linkage, based on Lander and Kruglyak (1995) criteria, tomarkers on Xq28 (Hamer et al. 1993). Another study,from the same laboratory but with a new sample, reported a significant replication of these findings (Huet al. 1995). An independent group produced inconclusive results regarding linkage to Xq28 (discussed inSanders and Dawood 2003) but did not publish thefindings in a peer-reviewed journal. All three of thesestudies excluded families showing evidence for nonmaternal transmission. A fourth study from anotherindependent group found no support for linkage, evenwhen excluding cases with suggestive father-to-sontransmission (Rice et al. 1999b). An analysis of the results across all four studies produced a statisticallysuggestive multiple scan probability (MSP) value of0.00003 (Sanders and Dawood 2003). Two candidategene studies have been conducted, both producing nullresults: one for the androgen receptor (AR; Macke et al.1993) and another for aromatase (CYP19A1; Dupreeet al. 2004), on Xq12 and 15q21.2, respectively.Given the complexity of sexual orientation, numerousgenes are likely to be involved, many of which are expected to be autosomal rather than sex-linked. Indeed,the modest levels of linkage that have been reported forthe X chromosome can account for, at most, only afraction of the overall heritability of male sexual orientation as deduced from twin studies. Therefore, we haveundertaken a genomewide linkage scan to aid in theidentification of genes contributing to variation in sexualorientation. As in previous studies, we diminished theprobability of false positives (i.e., gay men who identifyas heterosexual) by only studying self-identified gaymen. Unlike previous studies that have focused solely onthe X-chromosome and thus excluded families showingevidence of non-maternal transmission, this study didnot use transmission pattern as an exclusion criteria. Toconsider the possibility that previously reported evidenceof maternal loading of transmission of sexual orientation was attributable to epigenetic factors acting onautosomal genes, we calculated maximum likelihoodestimations (mlod) scores separated by maternal orpaternal transmission and the combined statistic. Basedon Lander and Kruglyak’s (1995) criteria, we found oneregion of near significance and two regions close to thecriteria for suggestive linkage.Materials and methodsFamily ascertainment and assessmentThe sample consisted of a total of 456 individuals from146 unrelated families, of which 137 families had two gaybrothers and 9 families had three gay brothers. Thirty ofthe families included one parent, and 30 of the familiesincluded both parents. Additionally, 46 of the familiesincluded at least one heterosexual male or female fullsibling (up to 6 additional siblings per family). Thesample included 40 families previously reported by Hamer et al. (1993), 33 families previously reported by Huet al. (1995), and 73 previously unreported families. The73 previously described families were selected for thepresence of two gay brothers with no indication of nonmaternal transmission by the criteria described previously (Hamer et al. 1993; Hu et al. 1995). For the 73 newfamilies, the sole inclusion criterion was the presence ofat least two self-acknowledged gay male siblings.Subjects were recruited through advertisements inlocal and national homophile publications as describedelsewhere (Hamer et al. 1993; Hu et al. 1995). The participants were predominantly white (94.5%), collegeeducated (87.4%), and of middle to upper socioeconomic status. The mean (SD) age for the gay siblingswas 36.98 (8.64). The protocol was approved by the NCIInstitutional Review Board, and each participant signedan informed consent form prior to interview, questionnaire completion, and the donation of blood for DNAextraction.Sexual orientation was assessed through a structuredinterview or a questionnaire that included a sexual history and the Kinsey scales of sexual attraction, fantasy,behavior, and self-identification (Kinsey et al. 1948).Each scale ranges from 0 (exclusively heterosexual) to 6
274Fig. 1 Genome scan results.The x-axis is the chromosomelocation (cM), and the y-axis isthe mlod score. Graphicsincluded for combined (a),maternal (b), and paternal (c)meioses(exclusively homosexual). The mean (SD) of these fourscales for the gay males in this study was 5.65 (0.46).GenotypingDNA was extracted from peripheral blood by a commercial service (Genetic Design, Greensboro, N.C.,USA). A multiplex polymerase chain reaction (PCR)was conducted as described (Dupree et al. 2004), with403 microsatellite markers from the ABI PRISM Linkage Mapping Set Version 2.5 with an average resolutionof 10 cM. Following the manufacturer’s guidelines,products were analyzed on an ABI Prism 310 or 3100and sized with the GeneScan version 3.1.2 program (PEBiosystems, Foster City, Calif., USA), and genotypeswere assigned with the Genotyper version 3.6 program(PE Biosystems). A PCR product from a DNA referencesample (CEPH 1347-02) was used to monitor sizingconformity (PE Biosystems). Across the 403 markers,genotypes were ascertained on average for 95% of the456 individuals. Mendelian incompatibilities ( 0.05%of genotypes) were removed from the data prior toanalyses by using the sib clean routine from ASPEXversion 2.4 (Hinds and Risch 1996). The computerprogram CERVUS 2.0 (Marshall et al. 1998) was em-
275ployed to test for deviation from the Hardy-Weinbergequilibrium (HW) and to calculate polymorphisminformation contents (PICs) at all loci. We found thatthe markers had a mean (SD) PIC of 0.76 (0.08), and1.31% of the markers deviated significantly from HW.Statistical analysesNonparametric exclusion mapping of affected sib-pairdata (ASP) was performed by using ASPEX version 2.4(Hinds and Risch 1996). ASPEX calculates the percentage of identical by descent (%IBD) sharing and reports the proportion of shared alleles of paternal,maternal, and combined origin. The results for alleles ofcombined origin also include alleles where the parentalorigin is unknown. We calculated mlod with a linearmodel and assuming a multiplicative model. TheASPEX SIB PHASE algorithm was applied; this usesallele frequency information to reconstruct and to phasemissing parental information. Sex-specific recombination maps were used for the calculation of multipointmlod scores. Marker order and map positions weredetermined by using an integrated map (Nievergelt et al.2004) based on the deCODE genetic map and updatedphysical map information.ResultsResults from the multipoint analyses on chromosomes 1through 22 are shown in Fig. 1 for paternal, maternal,and combined meioses. Our complete genome scan formale sexual orientation yielded three interesting peakswith mlod scores greater than 1.8, located on chromosomes 7, 8, and 10. Table 1 contains additional information concerning these peaks, including the nearestmarker, the location, MLOD, and allele sharing. Additionally, Table 1 contains the approximate boundary ofthe linkage peak, by reporting the approximate cM position at which the mlod score declines below 1.0. Forchromosomes 7 and 8, the peak is a result of approximately equal contributions from maternal and paternaltransmission, whereas a maternal-origin effect wasfound for the peak on chromosome 10.Figure 2 shows the multipoint mlod plots for the Xchromosome. Analyses of the full sample (dashed line)Fig. 2 Multipoint linkage analysis for the X chromosome. The xaxis is the chromosome location (cM), and the y-axis is the mlodscore. —— Current markers with sample restricted to previouslyreported families. - - - - Current markers with full sample. .Previously reported markers with previously reported familiesdid not produce any chromosomal regions with mlodscores greater than 1.0. Given the previous evidence oflinkage to Xq28 with a portion of the sample reportedhere (Hamer et al. 1993; Hu et al. 1995), we performedsupplemental analyses to determine why we did not findlinkage in the full sample. We began by re-analyzing thedata from the previously reported 73 families, which hadbeen selected for showing no evidence of paternaltransmission, by using updated marker positions (dottedline). This produced a maximum mlod score of 6.47 formarkers in the Xq28 region. We then performed alinkage analysis, with only the markers from the ABIlinkage mapping set, on these same 73 families. Thisproduced a maximum mlod score of 1.99 for markers inthe Xq28 region. Although the mlod score is higherwhen using the current markers in the limited samplecompared with the full sample (1.99 vs. 0.35), it is stillsignificantly lower than the previously reported markersin the limited sample. We provide Table 2 in order tohelp clarify these results. Table 2 provides singlepointand multipoint results for the 73 previously reportedfamilies on all markers ever reported from our group,starting with the most telemeric new Xq28 marker.Table 1 Chromosomal locations with nominally significant linkage peaks. The cM positions in parentheses indicate the boundary at whichthe mlod score declines below 1.0. For chromosomes 7 and 8, the position is based on the combined map, but for chromosome 10, theposition is based on the female mapNearby markerD7S798D8S505D10S217LocationmlodPercentageof sharingcMCytoPaternalMaternalCombined169.9 (155.1–end)54.2 (45.1–64.8)208.1 893.451.961.4362.5960.1058.51
276Table 2 Supplemental analyses comparing Xq28 results acrossmarkers reported on in 1995, 1997, and the current report. Allanalyses reported here are based on the sample restricted to thosefamilies previously reported. Current markers and previously reported markers were analyzed separately for the purpose of calculating multipoint mlod scoresMarkerStudy yearLocation(cM)Marker distance(cM)Multipoint mlod(previous .150.326.476.276.285.71Table 2 makes it clear that, although the multipointresults suggest a dramatic change in mlod score betweenthe current markers and the previously reported markers(6.47 vs. 1.99 for markers 0.62 cM apart), the singlepoint results are not dramatically different (2.23 vs.1.47). This difference is likely to be attributable to twofactors. First, the previous reports focused on the Xchromosome and contained many more markers in theXq28 region; the previously reported markers had anaverage resolution of 1 marker every 1.12 cM, whereasthe current markers had an average resolution 6.97 cMin the Xq28 region. The higher concentration of previously reported markers surely allowed for the extractionof more multipoint linkage information. Second, therewere more telomeric markers in the previously reportedmapping sets than in the current one. The singlepointresults showed a trend for higher mlod scores closer tothe telomere, with the exception of JXYQ28, which hada low PIC (0.28).DiscussionThis study reports results from the first full genome scanfor male sexual orientation. Using 73 previously reported families and 73 new families with two or moregay male siblings, we found three new regions of geneticinterest. Our strongest finding was on 7q36 with acombined mlod score of 3.45 and equal contributionfrom maternal and paternal allele transmission. Thisscore falls just short of Lander and Kruglyak’s (1995)criteria for genomewide significance. Several interestingcandidate genes map to this region of chromosome 7.Vasoactive intestinal peptide (VIP) receptor type 2(VIPR2; MIM 601970) is a G protein-coupled receptorthat activates adenylate cyclase in response to VIP(Metwali et al. 1996), which functions as a neurotransmitter and as a neuroendocrine hormone. VIPR2 isessential for the development of the hypothalamic suprachiasmatic nucleus in mice (Harmar et al. 2002), whichmakes it an interesting candidate gene for sexual orientation in view of earlier reports of an enlarged suprachiasmatic nucleus in homosexual men (Swaab andHofman 1990). Sonic hedgehog (SHH; MIM 600725)plays an essential role in patterning the early embryo,including hemisphere separation (Roessler et al. 1996)and left to right asymmetry (Tsukui et al. 1999).Multipoint mlod(current osexual men and women show a significant increasein non-righthandedness, which is related to brainasymmetry (Lalumiere et al. 2000).Two additional regions approached the criteria forsuggestive linkage. The region near 8p12 contains several interesting candidate genes, given the hypothesizedrelationship between prenatal hormones and sexualorientation (Mustanski et al. 2002). Gonadotropinreleasing hormone 1 (GNRH1; MIM 152760) stimulatesboth the synthesis and release of luteinizing hormoneand follicle-stimulating hormone, which are importantregulators of steroidogenesis in the gonads, and inhibitsthe release of prolactin (Adelman et al. 1986). GnRH issynthesized in the arcuate nucleus and other nuclei of thehypothalamus (Kawakami et al. 1975). Steroidogenicacute regulatory protein (STAR; MIM 600617) mediatespregnenolone synthesis and is involved in the hypothalamic-pituitary regulation of adrenal steroid production(Sugawara et al. 1995), which in turn plays an importantrole in sexual development. Neuregulin1 (NRG1; MIM142445) produces a variety of isoforms that regulate thegrowth and differentiation of neuronal and glial cellsthrough interaction with ERBB receptors (Burden andYarden 1997; Wen et al. 1994).The 10q26 region is of special interest because it results from excess sharing of maternal but not paternalalleles. Previous studies have suggested that there is anexcess of homosexual family members related to theproband through the mother, and we have proposedpreviously that this might result in part from genomicimprinting (Bocklandt and Hamer 2003). In support of aconnection between 10q26 and imprinting, a germlinedifferentially methylated region has been identified atthis location by Strichman-Almashanu et al. (2002) whoperformed a genomewide screen for normally methylated CpG islands and found 12 regions to be differentially methylated in uniparental tissues of germlineorigin, i.e., hydatidiform moles (paternal origin) andcomplete ovarian teratomas (maternal origin). SuchCpG islands can regulate the expression of imprintedgenes over distances of several hundred kilobases. Theregion ar
Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, Calif., USA Hum Genet (2005) 116: 272–278 DOI 10.1007/s00439-004-1241-4. related traits expressed at an early age (Bailey and
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