We detected several promising regions of multiple SNPs in the 10−5–10−7 p-value range, as seen in the Manhattan plot (Fig. 1), though no SNP reached genome-wide significance (5 × 10−8). The most prominent of these regions were on chromosomes 13 (minimum p = 7.5 × 10−7, rs9547443) and 14 (p = 4.7 × 10−7, rs1035144), where some SNPs had 10−7 < p < 10−6 (each region with 9 to 10 SNPs with p < 10−5, Table S1). There are a number of genes of relevance to the trait in and around these regions, which we describe below. We further note that the most significant SNP (rs77013977, p = 7.1 × 10−8) in the 23andMe male GWAS29 was nominally associated (p = 4.1 × 10−3) in our own GWAS. We used a meta-analytic statistic that did not need direction of effect, Fisher’s combined probability test30, which yielded p = 6.7 × 10−9 for this SNP, which is the first reported genome-wide significant association for the trait. As previously noted29, rs77013977 is an intronic SNP in NKAIN3, which is one of a family of four proteins (NKAIN1–4) suggested to be critical for neuronal function31.

Figure 1 Manhattan Plot of GWAS for Male Sexual Orientation. Plot of negative log 10 of the p-values for the single SNP association analysis of 1,077 homosexual and 1,231 heterosexual men, ordered along the x-axis for each chromosome by chromosomal position. Full size image

The strongest associated region on chromosome 13 (rs9547443, p = 7.5 × 10−7) was located between SLITRK6 (SLIT and NTRK like family member 6, ~60 kb centromeric to region) and SLITRK5 (~1.8 Mb telomeric), with SLITRK1 located ~2.0 Mb centromeric. Members of the SLITRK protein family are brain-expressed neuronal transmembrane proteins that regulate neuronal outgrowth, survival, and synapse formation; SLITRKs have significant homology to the secreted axonal growth-controlling SLIT family of proteins and also homology to the neurotrophic tyrosine kinase receptor (NTRK) family32,33,34. SLITRK6 is expressed especially in the diencephalon (which contains a region previously reported as differing in size in men by sexual orientation35), and SLITRK1 and SLITRK5 have their highest expression in the cerebral cortex32,33,34. Gene families, such as the SLITRK family that are important for neurodevelopment and are implicated as candidate genes for various neuropsychiatric phenotypes34, are also of potential relevance to behavioral phenotypes such as sexual orientation.

On chromosome 14, TSHR (thyroid stimulating hormone receptor) spans the region around our most significant SNP (rs1035144, p = 4.7 × 10−7), and includes a cluster of SNPs with association p < 10−5 in intron 1. TSHR encodes a G protein-coupled transmembrane receptor for thyrothropin (thyroid stimulating hormone) and thyrostimulin, manifests some constitutive activity (i.e., ligand independent), and is a major controller of thyroid cell metabolism36,37,38. While the main tissue of interest and expression for TSHR is the thyroid gland, TSHR is expressed in other tissues including brain especially in neuron-rich areas (e.g., hippocampus)39. TSHR codes for the major autoantigen in the autoimmune hyperthyroidism of Graves’ disease, which is associated (p < 10−20 with OR’s 1.4~1.5) with intron 1 polymorphisms40,41,42,43,44,45,46,47,48. A recent population-based study found that 5,351 same-sex married men among the assayed population of 2,252,751 Danish men had an elevated rate ratio of Graves’ disease (RR = 1.88; 95% CI = 1.08–3.01), a finding which held when excluding men with HIV/AIDS49. The authors49 speculate on the possibility that a genetic (or other prenatal) factor might tie together this increased risk for a type of hyperthyroidism (Graves’ disease) with separate observations of lower body weight for homosexual versus heterosexual men (independent of diet or exercise)50,51,52. Females with Graves’ disease have been reported to manifest biased X chromosome inactivation53,54,55, and skewed X chromosome inactivation has also been reported in mothers of homosexual men compared to age-matched mothers of heterosexual men56. Furthermore, a recent retrospective chart review of 790 adolescents (8 to 17 years) previously admitted to a child psychiatry service found 15 mothers with a history of thyroid dysfunction during pregnancy, 16 adolescents with a history of same-sex attraction and/or gender nonconformity, and 12 overlapping mother-offspring pairs with both (p < 0.0001), suggestive of a possible relationship57. Thus converging findings, including suggestive evidence from the current study, point to a possible connection between thyroid function and sexual orientation in men.

The main limitations of the current study include an exclusive focus on males, sampling primarily from one ancestral group (European), combination of two datasets, and most notably the modest sample size for a GWAS on a trait with complex genetics. Additional and larger sample sizes would be required to assess which loci might breach genome-wide significance for association in a single study, and to increase the number of such loci (as typically is the case with phenotypes with manifesting complex genetics58,59). Nevertheless, our study provides support for the top finding from a previous meeting report of a GWAS on the trait29, reaching genome-wide significance for the combined analysis of rs77013977 (p = 6.7 × 10−9) on pericentromeric chromosome 8. In addition, the current study’s top two association peaks (p < 10−5; Fig. 1) provide interesting and perhaps trait-relevant examples of their closest genes on chromosomes 13 (SLITRK6) and 14 (TSHR), though these potential connections are best characterized as speculative. The continued genetic study of male sexual orientation should help open a gateway to other studies focusing on genetic and environmental mechanisms of sexual orientation and development. Detectable genetic variants predisposing to homosexuality would have alternative alleles, which would necessarily predispose to heterosexuality, thus contributing to understanding of both typical heterosexual and minority homosexual orientations.