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CLINICAL CASE SEMINAR |
Unidade de Endocrinologia do Desenvolvimento, Laboratório de Hormônios e Genética Molecular LIM/42, Divisão de Endocrinologia, Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo (R.M.M., C.J.L., M.Y.N., A.E.C.B., A.C.L., B.B.M.), 01065-970 São Paulo, Brasil; and Department of Molecular Genetics, University of Texas Southwestern Medical Center (R.M.M., D.W.R.), Dallas, Texas 75390
Address all correspondence and requests for reprints to: Dr. Regina M. Martin, Divisão de Endocrinologia, Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo, Avenue Dr. Enéas de Carvalho Aguiar, 155 2°andar, Bloco 6, CEP 05403900, 01065-970 São Paulo, Brasil. E-mail: reginamm{at}usp.br.
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Abstract |
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-reductase type 1 also exhibit hyperestrogenism due to an increased availability of androgen precursors. Here we studied two adult siblings, born to unrelated parents, who presented clinical and hormonal evidence of estrogen excess. The man was treated with topical dihydrotestosterone, which promoted adequate virilization. The woman was treated with anastrazole, a potent aromatase inhibitor, with normalization of menstrual cycles. Genetic linkage to the steroid 5-reductase type 1 gene (SRD5A1) was ruled out in this family. A similar analysis did not rule out linkage to CYP19, although no mutation was identified in the coding region of this gene. Aromatase mRNA was at least 10-fold more abundant in the female patient’s skin fibroblasts vs. the control. Southern analysis of genomic DNA did not reveal rearrangements or amplification of the coding region of CYP19. We conclude that the phenotype of familial hyperestrogenism includes prepubertal gynecomastia, hypogonadism, and short stature in men, and precocious thelarche, macromastia, enlarged uterus, and menstrual irregularities in women. Topical dihydrotestosterone is an efficient alternative treatment in men with hyperestrogenism; in addition, second generation aromatase inhibitors are useful in both sexes. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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4A), testosterone (T), and 16-hydroxyandrostenedione, are, respectively, converted to estrone (E1), estradiol (E2), and estriol (E3) by P450 aromatase, the product of CYP19. Aromatase is the key enzyme in estrogen biosynthesis, which, together with the flavoprotein NADPH-P450 reductase, catalyzes the conversion of androgens to estrogens. Aromatase, like other heme-binding cytochrome P450 isoforms, is a highly conserved enzyme that is expressed in a complex, tissue-specific manner (1).
Human CYP19 contains 10 exons and is located on chromosome 15q21. It is expressed in the placenta, ovary, testis, brain, skin fibroblasts, adipocytes, normal breast and breast cancer epithelial and stromal cells, and a number of fetal tissues, including liver, brain, and intestine (2). The aromatase gene (CYP19) spans approximately 123 kb (3). The 5'-flanking region of the gene contains several different promoters that regulate transcription of CYP19 in different cell types (4), but all transcribed mRNAs specify an enzyme of 503 amino acids (5) (Fig. 1
). Tissue-specific regulation of CYP19 mRNA is accomplished in part by alternative splicing of exon 1 and part of exon 2 and is driven by at least seven promoters located in the 5'-flanking region of the gene (6, 7, 8, 9). Both enhancer and silencer sequences as well as binding sites for a variety of transcription factors have been identified in the regulatory regions of CYP19. Because of the complexity of its regulation, the 5'-end of CYP19 has been considered a candidate for disorders in which inappropriately high aromatase activity is encountered (2).
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Mice deficient in steroid 5-reductase type 1 have excess levels of estrogens and a deficient metabolism of progesterone in the uterine cervix (15, 16). These phenomena are credited to the increased availability of aromatizable androgens due to the loss of 5-reductase enzymatic activity. Normalization of estrogen levels and a waning of symptoms occur in these mice when an aromatase inhibitor or estrogen receptor antagonists are administered. The role of human 5-reductase type 1 is not understood, as mutations have yet to be described. Based on the phenotype of knockout mice, the loss of enzymatic activity of SRD5A1 could lead to reduced conversion of T to dihydrotestosterone (DHT) and subsequent accumulation of estrogen precursors.
A second animal model of hyperestrogenism is represented by Sebright Bantam and Campine chickens. In these strains the henny-feathering trait is a result of aromatase excess in the skin, which is caused by the integration of a proviral DNA into the aromatase gene promoter (17, 18, 19).
In the current paper comprehensive clinical, hormonal, and molecular data are provided for two Brazilian siblings with hyperestrogenism due to aromatase excess. In addition, a comparative analysis of these data and those previously described in four families with AES provides important insights into the pituitary-gonadal axis and alternative endocrine therapies for this condition.
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Subjects and Methods |
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The patients.
This study was approved by the ethics committee of Hospital das Clínicas, University of São Paulo, and informed written consent was obtained from both patients and their relatives for the clinical and laboratory studies. We studied two Brazilian Caucasian siblings born to nonconsanguineous parents. The proband presented prepubertal gynecomastia at 8 yr of age, reaching Tanner stage V when he was initially seen at age 13 yr with a bone age of 17 yr. He later underwent bilateral mastectomy. Physical examination at 21 yr of age showed: height, 161.3 cm (-1.58 SD), which was lower than his target height of 169 cm (-0.45 SD); weight, 57 kg; small penis size, 6.5 x 2.0 cm (-4.25 SD); scarce body and pubic hair; and gynecoid fat distribution (Fig. 2). A sperm count disclosed a low number of immobile sperm cells (6 million/ml). Testis ultrasonography and adrenal computed tomography scan did not show alterations suggestive of an estrogen-producing tumor. The patient had a normal 46,XY karyotype.
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Basal hormone levels.
LH, FSH, T, and E2 were measured by immunofluorometric assays, and DHT, 4A, and E1 were measured by RIA in both patients (20) (Tables 1 and 2).
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and 2). The results were compared with normal values established in our laboratory (21, 22, 23). Clinical evaluation and treatment
The male patient was treated with topical DHT, a nonaromatizable androgen (
100 mg/d) at 21 yr of age, with a consequent increase in body hair and penis size (9.2 x 2.0 cm; -2.4 SD) 1 yr after the treatment was started.
His sister was treated initially with progesterone (nomegestrole, 5 mg/d) to antagonize the high levels of estrogens, and required 21 d of oral progesterone intake to decrease menstrual flow. Subsequently, nomegestrole was given with anastrozole (2 mg/d) continuously to normalize menstrual periods. Later, progestagen was discontinued and her clinical, hormonal, and imaging parameters remained under control. The uterine volume, after 3.5 months of treatment with 2 mg/d anastrozole alone, decreased to 97 cm3, and endometrial thickness remained within the normal range. The patient has undergone elective breast reduction, and enlargement of the breast did not recur during anastrozole treatment.
Molecular analysis
Amplification and direct sequencing of genomic DNA. Genomic DNA was obtained from peripheral blood leukocytes using a salt precipitation method. The coding regions of the SRD5A1 and CYP19 genes were amplified by PCR using previously described intronic primers for the former (24) and designed intronic primers for the latter (25). The first exon of SRD5A1, which is rich in GC, was amplified after an extended initial denaturation step (95 C for 5 min), followed by 30 cycles of denaturation (95 C for 50 sec), annealing (57 C for 50 sec), and extension (72 C for 50 sec), followed by a final extension step at 72 C for 5 min in a thermal cycler (PE 9700, Perkin-Elmer, Foster City, CA). Dimethylsulfoxide was added (10%, vol/vol) to the 1x PCR buffer [20 mM Tris-HCl (pH 8.4) and 50 mM KCl], 1.5 mM MgCl2, 0.2 mM deoxynucleoside triphosphate, 10 pmol of each primer, and 2.5 U Platinum Taq DNA polymerase (Life Technologies, Inc., Gaithersburg, MD). Exons 2–5 of the SRD5A1 gene were amplified by PCR with the following conditions: 35 cycles of denaturation (94 C for 30 sec), annealing (55 C for 15 sec), and extension (72 C for 60 sec). Exons II–X of CYP19 were amplified by 30 cycles of denaturation (94 C for 30 sec), annealing (57 C for 30 sec), and extension (65 C for 30 sec). All PCR products were sequenced directly using an automated fluorescence-based dideoxynucleotide termination method (ABI 310, PE Applied Biosystems, Foster City, CA).
CYP19 linkage analysis. A polymorphic marker composed of varying numbers of tetranucleotide repeats located within human CYP19 on chromosome 15q21.1 (26) and two additional microsatellites (D15S209 and D15S1016) located within a genetic distance less than 5 cM from the CYP19 were scored (14). These regions were amplified from genomic DNA using primers and conditions described previously. The fragment containing the intragenic marker was sequenced to define the number of TTTA repeats for each allele. After PCR amplification of the two microsatellites, DNA fragments were resolved by electrophoresis on a 6% denaturing polyacrylamide gels and visualized by silver staining. For microsatellites D15S1016 and D15S209, the shortest allelic variant was arbitrarily designated A, and the other alleles were denominated B, C, and D according to crescent size.
Southern analysis. Genomic DNA was prepared from the peripheral blood leukocytes of patients, their mother, and a normal control. Southern blotting of DNA samples (digested with five enzymes: EcoRI, HindIII, BamHI, XbaI, and KpnI) were followed by hybridization with an aromatase cDNA probe spanning all of the coding exons of the gene (27). A control probe consisting of exons 7–8 of CYP17 was used to judge amplification.
Skin fibroblast culture, RNA extraction, and semiquantitative RT-PCR. A skin biopsy from the dorsal forearm area was obtained under local anesthesia from the female patient and from the first author of this study who served as the normal control. Fibroblasts were grown by standard methods. Total RNA was prepared from fibroblasts using TRIzol (Life Technologies, Inc.) (28). After treatment of the RNA with deoxyribonuclease I (1 U deoxyribonuclease I/µg total RNA; Life Technologies, Inc.), 1.7 µg total RNA from the patient’s cells and a similar amount from the control were used as a template for cDNA synthesis using 200 U Superscript II reverse transcriptase (Life Technologies, Inc.) and 1 µl oligo(deoxythymidine)12–18 (Life Technologies, Inc.). An incubation of 20 min at 37 C was performed with 2 U ribonuclease H (Life Technologies, Inc.) to degrade template RNA.
CYP19 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs were coamplified using two pairs of primers (29, 30). The amplification protocol consisted of 35 cycles of denaturation (94 C for 35 sec), annealing (55 C for 50 sec), and extension (72 C for 90 sec), preceded by an initial denaturation step of 94 C for 3 min and followed by a final extension step of 72 C for 5 min. Arbitrarily, the forward primers were radiolabeled with 32P, and different volumes (1, 3, and 10 µl) of cDNA from each sample were used as templates for the amplification reactions. After electrophoresis on 8% nondenaturing polyacrylamide gels, DNA fragments were visualized by ethidium bromide staining. Each CYP19 and GAPDH DNA was excised from the gel and mixed with 500 µl NCS II tissue solubilizer (Amersham Pharmacia Biotech, Arlington Heights, IL) and 5 ml scintillation cocktail. After 24 h, the radioactivity of each DNA fragment was measured by scintillation counting. The CYP19 signal from each reaction was normalized to the signal from the corresponding GAPDH signal.
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Results |
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The male patient at 21 yr of age had high levels of both E1 and E2, whereas his T and FSH levels were decreased. After acute GnRH stimulation, a pubertal LH peak was observed that was lower than adult levels. FSH levels were suppressed, probably by the high estrogen levels. After human chorionic gonadotropin testing, an increase in T levels was observed, followed by a remarkable elevation of estrogen levels. T injection resulted in a huge elevation of E2 levels, indicating an important contribution of extragonadal tissues to excess aromatization. After 1 wk of oral intake of anastrozole, T, E1, E2, and gonadotropin levels returned to normal (Table 1). The female patient at 22 yr of age had high levels of E1 and E2, nonovulatory levels of progesterone, and normal LH and FSH levels after a GnRH test (Table 2). After 1 wk of anastrozole treatment, her estrogen levels were normal, and weekly measurements during therapy showed ovulatory levels of progesterone.
5-Reductase type 1 gene analysis
The entire coding region of the SRD5A1 (exons 1–5) was amplified and sequenced. No mutations were found upon comparison with the published sequence of the normal human gene (24, 31), although known polymorphisms in codons 30, 103, 116, and 160 were identified in the heterozygous state. Segregation analysis of these polymorphisms ruled out SRD5A1 as the cause of the hyperestrogenic syndrome, because the affected siblings did not share the same haplotypes (data not shown).
CYP19 linkage analysis
The number of TTTA tetranucleotide repeats ranged from seven to eight, whereas CA repeats ranged from A to D and A to C for microsatellites D15S1016 and D15S209, respectively. The affected siblings share the same maternal and paternal haplotypes, different from the nonaffected brother, permitting linkage between CYP19 and the estrogen excess condition (Fig. 3). The 7DB haplotype shared by the two affected siblings, but absent in the normal brother, may suggest autosomal dominant inheritance with incomplete penetrance, as the mother does not show clinical or hormonal evidence of this syndrome. However, we cannot rule out the possibility of an autosomal recessive inheritance.
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Coding region. Exons II–X of CYP19 were amplified and found to have the expected sizes. Direct sequencing of the nine DNA fragments revealed no mutations in the coding region of the estrogen excess patients.
Genomic Southern analysis. The patients’ DNA showed the same hybridization pattern as that of the mother and a normal control DNA, which excludes an intragenic or major rearrangement in the CYP19 as cause of the disease. A control probe consisting of the coding region of CYP17 showed a signal of similar hybridization intensity, which excluded genetic amplification of CYP19 as the molecular basis of the syndrome.
Semiquantitative RT-PCR from skin fibroblast culture (female patient and control).
Although the aromatase cDNA fragments showed exponential amplification in aliquots of 1, 3, and 10 µl from the control, samples from the female patient presented maximum amplification with minimal volumes of input cDNA (Fig. 4). The ratio of GAPDH/CYP19 cDNA fragments for each sample revealed that aromatase mRNA expression was at least 10 times greater in the female patient than in the control.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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). In this rare condition, of the 12 affected male patients mentioned, all of them developed prepubertal (50%) or peripubertal (50%) gynecomastia, and short stature was present in 4 of 6 patients evaluated through adulthood (Table 3). In the current report we studied a Brazilian family in which the proband developed prepubertal gynecomastia associated with advanced bone age, compromised final height (32), micropenis (33), and hypogonadic signs, features recognized in this syndrome.
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The accelerated growth, advanced bone age, and compromised final height presented by these patients contrast with the tall stature of men with aromatase deficiency or estrogen resistance (34, 35, 36, 37), indicating that the effect of estrogens on bone maturation is not restricted to the female sex. Moreover, in both patients we identified an upper limit of bone density despite hypogonadism in the man and irregular menses in the woman.
Fertility is probably not always affected in AES, as we observed vertical transmission through both sexes. It was normal in two families reported in the literature, but was probably impaired in our patient considering the observed oligozoospermia. It is significant that the father of family 4 had small testes, as did our patient. In contrast, one man with severe aromatase deficiency had macroorchidism related to the high levels of gonadotropins secondary to the loss of negative feedback by estrogens; estrogen replacement normalized the size of the testes in this individual (35). In humans, estrogen-secreting tumors or exogenous estrogen intake in male transsexuals decreases testis size and fertility. Furthermore, intratesticular T is needed for spermatogenesis induction in men (38). Therefore, we conclude that the spectrum of testis volume and fertility alterations present in aromatase excess depends on the degree of aromatization, which will directly impact the levels of gonadotropins and the production of T. The vertical transmission of the disorder by a paternal grandmother in family 4 suggests that fertility in females is not always affected. On the other hand, fertility may be altered in our female patient, based on her phenotype (irregular menses and anovulatory cycles).
Several lines of evidence suggest that E2 as well as T regulate gonadotropin production (34, 35, 36, 37, 38, 39, 40, 41). These data suggest that the low T levels in our male patient may have arisen due to estrogen-mediated suppression of GnRH with subsequent impairment of gonadotropin production. The suppressed FSH levels and the normal LH levels observed in our case after the GnRH test contrasted with data from the men of families 2 and 4, who presented with normal FSH levels. We attribute this result to a higher level of aromatization in our patient. LH and FSH levels were normal in the female patient despite the high estrogen levels and were comparable with those in the female patient of family 4.
In the present study both patients had significantly high basal levels of estrogens, particularly of E1, as observed in other patients (2, 13). The high levels of estrogens normalized with aromatase inhibitor therapy, showing that aromatization excess in vivo was the cause of hyperestrogenism. The ratio of E2/T after T injection in the male patient was 100 times greater than that in the control group, indicative of peripheral conversion of androgens into estrogens.
In the past the treatment of this syndrome was limited to radical mastectomy in men (10, 11), but aromatase inhibitor viability amplified the therapeutic resources (2, 13). Therapy with GnRH analogs when central precocious puberty is detected before patients reach final stature could also minimize stature loss (2). The hyperestrogenism in our female patient was controlled with daily anastrozole administration; the man, instead, was treated with DHT, which appears to be an effective and alternative treatment for this disorder in men. However, we do not know whether AES is associated with a greater prevalence of estrogen-depend tumors, as estrogen excess was not treated in the father of family 3, who had carcinoma of the breast (13).
In summary, we conclude that the phenotype of familial hyperestrogenism includes prepubertal gynecomastia, hypogonadism, and short stature in men, and precocious thelarche, macromastia, enlarged uterus, and menstrual irregularities in women. A good therapeutic response was obtained with DHT or anastrozole in an affected male and with anastrozole in affected female.
The analysis of the available pedigrees permits some considerations about the inheritance pattern of AES (Fig. 5). In family 2 (11), for example, the researchers suggested an X-linked or autosomal dominant inheritance with sex-limited expression, not considering the possibility of affected females, before the location of CYP19 was established. Nevertheless, after the description of families 3 and 4, autosomal dominant inheritance seems the most probable (2), especially considering that the disease spanned three generations, and a CYP19 polymorphism segregated with the AES in the family 4. In our family, as the two affected siblings share the same haplotypes, either an autosomal dominant with incomplete penetrance or an autosomal recessive inheritance pattern was considered (Fig. 2). Moreover, considering the different phenotypes of the AES patients, we cannot discard the possibility of variable expressivity of the CYP19 gene.
The literature provides little information about the molecular basis of AES. In family 4, aromatase enzyme activity (measured by [3H]4A to [3H]E1 conversion) and mRNA expression were increased in cultured skin fibroblasts and Epstein-Barr virus-transformed lymphocytes from affected family members. Illegitimate CYP19 transcripts were identified in these cells derived from affected patients, and they appeared to contain novel exon I sequences (2). DNA sequence analysis revealed that the abnormal mRNAs stemmed from promoter PII (according to the Simpson nomenclature adopted in this paper). The identification of a higher level of this aberrant mRNA in these cells as well as in breast tissue from the patients was proposed as an explanation for the excess aromatization in this family, but the mechanism leading to the use of the PII promoter was not identified.
Here, we tested the hypothesis that steroid 5-reductase type 1 contributed to the disorder based on excessive estrogen production observed in knockout mice for this gene (15, 16). This hypothesis was ruled out in our family because of the absence of cosegregation between phenotype and genotype. Although no mutations in the CYP19 coding region were found by DNA sequence analysis, further analysis of CYP19 did not exclude the involvement of this gene in the etiology of this disease in the family we studied. We also excluded major intragenic rearrangements as the cause of the disorder. Despite these negative data, aromatase expression in skin fibroblasts of the female patient was 10 times greater than that in a control fibroblast line, indicating that this tissue is in part responsible for the excessive aromatization. These data suggest that alterations in the CYP19 regulatory region may be responsible for overexpression of the gene, as reported previously (42, 43).
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Acknowledgments |
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Footnotes |
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Abbreviations: 4A, Androstenedione; AES, aromatase excess syndrome; DHT, dihydrotestosterone; E1, estrone; E2, estradiol; E3, estriol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; T, testosterone.
Received November 12, 2002.
Accepted March 31, 2003.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-reductase type 1 caused by estrogen excess. Mol Endocrinol 11:917–927-reductase type 1 knockout mice is due to impaired cervical ripening. Mol Endocrinol 13:981–992-reductase 2 deficiency. Medicine 75:64–76[CrossRef][Medline]
-reductase gene and pseudogene and mapping of the mouse homologue. Genomics 11:1102–1112[CrossRef][Medline]
-reductase: two genes/two enzymes. Annu Rev Biochem 63:25–61[Medline]
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