Genetic determinants of serum testosterone concentrations in men

Abstract

Testosterone concentrations in men are associated with cardiovascular morbidity, osteoporosis, and mortality and are affected by age, smoking, and obesity. Because of serum testosterone's high heritability, we performed a meta-analysis of genome-wide association data in 8,938 men from seven cohorts and followed up the genome-wide significant findings in one in silico (n = 871) and two de novo replication cohorts (n = 4,620) to identify genetic loci significantly associated with serum testosterone concentration in men. All these loci were also associated with low serum testosterone concentration defined as <300 ng/dl. Two single-nucleotide polymorphisms at the sex hormone-binding globulin (SHBG) locus (17p13-p12) were identified as independently associated with serum testosterone concentration (rs12150660, p = 1.2×10(-41) and rs6258, p = 2.3×10(-22)). Subjects with ≥ 3 risk alleles of these variants had 6.5-fold higher risk of having low serum testosterone than subjects with no risk allele. The rs5934505 polymorphism near FAM9B on the X chromosome was also associated with testosterone concentrations (p = 5.6×10(-16)). The rs6258 polymorphism in exon 4 of SHBG affected SHBG's affinity for binding testosterone and the measured free testosterone fraction (p<0.01). Genetic variants in the SHBG locus and on the X chromosome are associated with a substantial variation in testosterone concentrations and increased risk of low testosterone. rs6258 is the first reported SHBG polymorphism, which affects testosterone binding to SHBG and the free testosterone fraction and could therefore influence the calculation of free testosterone using law-of-mass-action equation.

Figure 1. Regional association plots for single-nucleotide polymorphisms rs12150660, rs6258, and rs5934505.

Regional association plot of the two independent signals on chromosome 17 with either (A) rs12150660 or (B) rs6258 indicated by red diamond to evaluate linkage with other single-nucleotide polymorphisms in the region. In addition, the association plot of the (C) rs5934505 signal on chromosome X is given. The r² is based on the CEU HapMap II samples. The blue line and right hand Y axis represent CEU HapMap II based recombination rates. (A) and (B) show the top SNPs of the inverse-variance weighted discovery stage meta-analysis of untransformed serum testosterone and (C) show the top SNP of the SHBG-adjusted serum testosterone using an imputation quality filter (observed/expected variance ratio) >0.4 at the individual cohort level during meta-analysis.

Figure 2. The genetic influence on low serum testosterone concentrations.

(A) Odds ratio (OR) for risk of low serum testosterone concentrations (serum testosterone <300 ng/dl), per copy of minor allele. Summary estimates of the OR and their 95% confidence intervals (CI) are given. The size of the data markers is proportional to the weight (inverse of the variance) of each study. Combined discovery (n = 8,030, low serum testosterone 13%; KORA was not included as testosterone was analyzed in plasma rather than in serum, combined replication (n = 5,504, low serum testosterone 13%), and combined discovery and replication (n = 13,534, low serum testosterone 13%). (B) Percentage of men with low serum testosterone concentrations (serum testosterone <300 ng/dl), according to the number of combined risk alleles for rs12150660 (G) and rs6258 (T) in the three replication cohorts (MrOS Sweden, EMAS, and YFS). Only two individuals in the three replication cohorts had four risk alleles and therefore individuals with three and four risk alleles were pooled into one group with ≥3 risk alleles.

Figure 3. SHBG affinity for testosterone.

(A and B) Scatchard plots of SHBG binding affinity for testosterone in serum samples according to rs6258 genotype. (A) Representative Scatchard plots of serum SHBG binding to [³H]testosterone. Serum from individuals homozygous for the wild-type SHBG allele (CC dashed line) or the rs6258 SNP (TT, solid line), or heterozygous for these alleles (CT, solid line). (B) Dissociation constant (Kd) of serum SHBG according to rs6258 genotype (CC, n = 4 subjects; CT, n = 4 subjects; TT [rare variant] n = 1 and the variation for the TT subject is derived from three separate analyses). (*) p = 0.001. Values are means ± SEM. (C) Representative Scatchard plots of recombinant SHBG binding to [³H]testosterone. Recombinant wild type ( = WT, C genotype; dashed line) or rs6258 (T genotype; solid line) SHBG expressed by CHO cells was diluted 1∶10 and subjected to Scatchard analysis, as in panel A. (D) Free testosterone fraction in serum measured by an equilibrium dialysis method according to rs6258 genotype (CC, n = 87 subjects; CT, n = 32 subjects). Values are means ± SEM.