A. Background and Clinical Manifestations
Wilson and McPhaul and their colleagues described a genetically inherited form of estrogen excess in henny-feathered Sebright roosters (George and Wilson, 1980; McPhaul et al., 1988; Matsumine et al., 1990). In birds, aromatase is normally expressed in the ovaries and brain but not in skin fibroblasts (McPhaul et al., 1988; Simpson et al., 2002). In Sebright chickens with the autosomal dominant henny-feathered trait, aromatase is overexpressed in skin fibroblasts, leading to inappropriate estrogen formation in skin and a female pattern of feather development in roosters (George and Wilson, 1980; Matsumine et al., 1990). Furthermore, the 5’-untranslated region of aromatase mRNA in skin fibroblasts suggested that a unique promoter regulated the expression of aromatase (Matsumine et al., 1990). We published two distinct rearrangements in chromosome 15, which may be responsible for the human counterpart of this disorder, i.e., aromatase excess syndrome (Shozu et al., 2003b)
Boys affected by this syndrome present with growth of breast tissue (gynecomastia), whereas girls present with premature breast development (thelarche). Both sexes exhibit a premature growth spurt, early fusion of epiphyses, and decreased adult height. Adult men are undervirilized, whereas adult women experience irregular uterine bleeding. Increased conversion of steroid precursors to estrogens in extraglandular tissues is responsible for excessive estrogen formation. MacDonald and his colleagues described originally a feminized, prepubertal adopted boy in whom large amounts of estrone and estradiol were produced by extraglandular aromatization of plasma androstenedione (Hemsell et al., 1977). Five families were subsequently described in which several members had estrogen excess as a result of increased extraglandular aromatization inherited in an autosomal dominant fashion (Berkovitz et al., 1985; Leiberman and Zachmann, 1992; Stratakis et al., 1998; Martin et al., 2003; Shozu et al., 2003b).
B. Genetic Basis for Familial Aromatase Excess Syndrome
We evaluated for genetic abnormalities a 36-year-old man, his 7-year-old son, and an unrelated 17-year-old boy with severe gynecomastia of prepubertal onset and hypogonadotropic hypogonadism caused by elevated estrogen levels (Shozu et al., 2003b). Aromatase activity and mRNA levels in fat and skin and conversion of circulating androstenedione to estrone were severely elevated. Treatment with an aromatase inhibitor decreased serum estrogen levels and normalized gonadotropin and testosterone levels. The 5’-untranslated regions of aromatase mRNA contained the same sequence (FLJ) in the father and son and another sequence (TMOD3) in the unrelated boy; neither sequence was found in control subjects nor had they been previously described (Fig. 20). These 5’-untranslated regions normally make up the first exons of two ubiquitously expressed genes clustered in chromosome 15q21.2 to 3 in the following order (from telomere to centromere): FLJ, TMOD3, and aromatase. The aromatase gene is normally transcribed in the direction opposite to that of TMOD3 and FLJ. Two distinct heterozygous inversions reversed the direction of the TMOD3 or FLJ promoter in the patients (Shozu et al., 2003b) (Fig. 20).
We found that overproduction of estrogen arose from gain-of-function mutations in chromosome 15, giving rise to the formation of cryptic promoters that regulate the aromatase gene. These constitutively active promoters normally serve to transcribe two ubiquitously expressed genes—FLJ and TMOD3—that encode products homologous to muscle proteins in many human tissues. The functions of FLJ and TMOD3 are not known (Cox and Zoghbi, 2000; Nagase et al., 2000) (Fig. 20). An identical mutation involving the FLJ promoter was transmitted from a father to his son in an autosomal-dominant manner (Fig. 20). The cryptic promoter in a third unrelated patient was distinct and arose from the TMOD3 gene (Fig. 20). The relatives of this third patient were not affected. Therefore, this rearrangement seemed to be the result of a new mutation.
These mutations are somewhat analogous to transgenic mice, in which a gene product fused to a constitutively active promoter is expressed in multiple tissues after random insertion. The tissue selectivity (or nonselectivity) and level of activity of this aberrant promoter determines the tissue distribution and levels of mRNA encoded by this gene. In human peripheral tissues and gonads, basal levels of aromatase mRNA is extremely low. Only FSH induces aromatase expression physiologically to very high levels in the ovaries of premenopausal women during cycle days 5 to 14. Thus, in general, low basal expression of aromatase is tightly controlled in the peripheral tissues because comparatively low levels of aromatase expression and formation of estrogen (measured in picomoles per liter in the serum) are sufficient for many estrogenic effects. The mutations described in aromatase excess syndrome represent a major deviation from this physiology and are caused by constitutively active cryptic promoters that take control over aromatase expression.
C. Discussion on Gain-of-Function Mutations That Affect the Aromatase Gene
The upstream region of the aromatase gene may represent a “hot spot” for mutations. Occasional mutations, such as those that we described, caused extremely high aromatase activity and striking clinical consequences. More common rearrangements, however, may go clinically unrecognized and cause subtle degrees of estrogen excess, which may increase the risks of estrogen-dependent disease, such as cancers of the breast and endometrium, endometriosis, and uterine fibroids.
Adult men with this syndrome had hypogonadotropic hypogonadism. During treatment with an aromatase inhibitor, their estrogen levels declined and testosterone, luteinizing hormone, and follicle-stimulating hormone levels rose to normal. This response suggests a crucial role of estrogen in the suppression of both gonadotropins in men. Despite low testosterone levels in these patients, luteinizing hormone remained suppressed, possibly owing to the high levels of circulating estrogen. These mutations may have given rise to the overexpression of aromatase in the brain and thus to increased local estrogen production, which might also have contributed to the suppression of gonadotropins.
Aromatase inhibitors may potentially be used to prevent or halt the progression of gynecomastia in young boys (Plourde et al., 2004). The potential objective of long-term treatment of adult men with an aromatase inhibitor is to restore gonadal function, but this approach is not clearly justified for several reasons (Shozu et al., 2003b). First, men with this condition are not at risk for osteoporosis. Second, although men with estrogen excess may be subfertile, infertility is not a uniform feature of the syndrome. Third, the consequences of long-term exposure to high levels of estrogen in men are not known. We suggest that these men should periodically be evaluated for breast and prostate disease, given the potentially deleterious effects of estrogen on these tissues (Shozu et al., 2003b; Plourde et al., 2004).
There are no published reports regarding the use of the new-generation aromatase inhibitors in women with aromatase excess syndrome. It is conceivable that these women are at increased risk for developing breast cancer, endometrial cancer, macromastia, endometriosis, and uterine fibroids. Thus, aromatase inhibitors may potentially be used for prevention or treatment. Answers to these questions require clinical trials in carefully selected populations.
VII. Conclusions Regarding Aromatase Overexpression in Estrogen-Dependent Human Disease
Several principles emerge from the data summarized in this review. First, human aromatase encoded by a single gene acts as a master switch in physiological and pathological systems to turn on estrogen formation or serves as a therapeutic target to turn off estrogen biosynthesis. By the same token, estradiol acts as a master switch in many biological systems to regulate hundreds to thousands of diverse genes at one time point, which makes it extremely difficult to therapeutically target any of these downstream genes.
Second, biologically active quantities of aromatase mRNA and protein and their product estrogen are relatively small. For example, circulating or tissue estradiol is measured in picomole quantities that are 100 to 1000 orders of magnitude smaller than those used for testosterone measured in nanomolar quantities. Thus, comparatively small levels of aromatase expression have physiologically or pathologically critical consequences.
Third, local aromatase expression and estrogen biosynthesis seems to be a far more critical therapeutic target compared with circulating estrogen. Fourth, complex and redundant epigenetic mechanisms are responsible for aromatase expression in the estrogen-dependent pathologic tissues. These range from accumulation of aromatase-expressing cell types to aberrant expression of transcriptional enhancers and permit the use of additional promoters in pathological tissues, which are normally silenced in disease-free tissue counterparts. Most notably, the two proximal promoters, I.3 and II, which are located within 215 bp from each other and coordinately regulated by PGE2 or protein kinase A- and C-dependent pathways, are aberrantly activated in breast cancer, endometriosis, endometrial cancer, and uterine fibroids.
Finally, gain-of-function-type germ cell mutations give rise to cryptic promoters that overexpress the aromatase gene ubiquitously in many tissues. We speculate that the promiscuous nature of the splice acceptor site immediately upstream of the coding region is in part responsible for regulation of the aromatase gene by multiple alternatively used physiological or pathological promoters.