Biomedical subjects
Publications and source records attributed to C J Migeon.
A diagnosis of androgen receptor deficiency was made in a male with ambiguous genitalia during the neonatal period. Since the neonate had a small hypospadiac phallus with laboratory evidence of a receptor deficiency and clinical unresponsiveness to high levels of androgen, it was decided to assign a female gender. Bilateral orchiectomy and phallic recession were performed.
We studied the binding of dihydrotestosterone-receptor complexes (DHT-R) from human genital skin fibroblasts to oligodeoxyribonucleotides and DNA. Following incubation of fibroblasts with 2 nM [3H]DHT (45 min, 37 degrees C), DHT-R were prepared as total fibroblast sonicates (sonication of cells in 0.5 M KCl), intact fibroblast cytosol (100000 X g supernatant) or intact fibroblast nuclear extract (sonication of nuclei in 0.5 M KCl). DHT-R were also prepared by incubation of fractionated fibroblast cytosol with 4 nM [3H]DHT (4 h, 0 degrees C). Optimal conditions were established for binding of DHT-R from total fibroblast sonicates to oligo-dT cellulose: 60 min, 0 degrees C, low salt (0.05-0.10 M KCl), linearity with DHT-R concentration, and nucleotide saturation. With total fibroblast sonicates the rank order of DHT-R binding was oligo-dT approximately equal to -dG greater than DNA greater than -dC greater than or equal to -dA approximately equal to -dI. Intact fibroblast cytosol displayed a similar preference of DHT-R binding to oligo-dT and -dG but the binding was quantitatively higher than for total fibroblast sonicates, the binding for fractionated fibroblast cytosolic DHT-R formed at 0 degrees C being quantitatively lower. However, binding of DHT-R from cytosol (0 degrees C) to DNA-cellulose was equal to that for DHT-R from cytosol (37 degrees C). Binding of DHT-R from intact fibroblast nuclear extracts was lower than for total fibroblast sonicates. Preparations from cells of patients with receptor-negative, complete androgen insensitivity lacked both DHT-R formation and specific oligonucleotide binding. Binding of oligonucleotides to DHT-R from cells of patients with receptor-positive, complete androgen insensitivity could not be distinguished from that of normal cells. These results suggest: (a) androgen receptor-steroid complexes recognize and bind to certain preferred deoxyribonucleotides; (b) various factors affect the quantitative binding of DHT-R from different cellular preparations to deoxyribonucleotides; and (c) neither qualitative nor quantitative abnormalities for DHT-R of complete androgen-insensitive patients were detectable from oligonucleotide or DNA binding.
Testosterone and its active metabolite dihydrotestosterone exert their influence on target cells through a specific intracellular protein receptor. Structural abnormalities of this receptor lead to a diminished androgen action within the cell and result in the syndrome of androgen insensitivity. Androgen insensitivity is classified on the basis of whether the insensitivity is complete or partial and whether the androgen receptor is normally present (AR(+)), absent (AR(-)) or diminished (AR(+/-)). All patients with androgen insensitivity have normal or high plasma levels of testosterone and elevated serum LH. Patients with complete androgen insensitivity are phenotypically female. The clinical presentation of partial androgen insensitivity is variable, ranging from a minimal amount of virilization to a completely masculine appearance. All patients described with a syndrome of androgen insensitivity are infertile. The influence of androgen receptor function in the pathogenesis of benign prostatic hypertrophy is being investigated. Androgen receptor content is also being studied as a possible marker of responsiveness to hormonal therapy in prostatic carcinoma.
In male pseudo-hermaphroditism due to end organ unresponsiveness, partial androgen insensitivity can usually be related to low but detectable dihydrotestosterone (DHT) binding activity in cultured sexual skin fibroblasts. We have studied two patients with partial virilization and total absence of androgen receptors in cultured fibroblasts. A lack of DHT binding could be related to a selection of a fibroblast strain with no androgen receptor activity, to insufficient sensitivity of the method in detecting very low receptor concentrations or to an instability of the receptor or the androgen-receptor complex. However, these observations raise the question of masculinization of external genitalia with undetectable androgen receptor.
A family with nine children, three with male pseudohermaphroditism due to testicular deficiency of 17-ketosteroid reductase activity (17-KSR) and four with congenital hypothyroidism is presented. The three subjects with 17-KSR deficiency were raised as females until puberty, at which time they assumed a male gender role. Only one developed gynecomastia. Laparotomy on one of the three patients revealed normal epididymi and vas deferens with absence of Mullerian structures. Testicular biopsy in all three showed Leydig cell hyperplasia, hyalinization of the tubular basement membrane, normal Sertoli cells and maturational arrest at the spermatogonial stage. The endocrine profile in peripheral blood revealed markedly increased plasma androstenedione concentrations but normal testosterone, dihydrotestosterone, progesterone, 17-hydroxyprogesterone, and dehydroepiandrosterone. The levels of estradiol and estrone and of LH and FSH were elevated. Genital skin fibroblasts from the three patients exhibited normal dihydrotestosterone-binding activity and 5 alpha-reductase activity. Congenital hypothyroidism affected one of the three siblings with male pseudohermaphroditism. All four hypothyroid patients had thyroid enlargement and significant titers of circulating antithyroglobulin but not antithyroid microsomal antibodies. Neither the locus for the 17-KSR enzyme nor that for congenital hypothyroidism were linked to the histocompatibility leucocyte antigen complex in this sibship. Transmission of the trait for both congenital hypothyroidism and 17-KSR deficiency appeared to be autosomal recessive.
Nuclear DNA from individuals belonging to nine different families in which two sibs were affected with isolated growth hormone deficiency type I were studied by restriction endonuclease analysis. By using 32P-labeled human growth hormone or the homologous human chorionic somatomammotropin complementary DNA (cDNA) sequences as a probe, the growth hormone genes of affected individuals from all families yielded normal restriction patterns. Polymorphic restriction endonuclease sites (HincII and MspI), which are closely linked to the structural gene for growth hormone on chromosome 17, were used as markers in linkage analysis of DNA of family members. Of the nine affected sib pairs two were concordant, three were possibly concordant, and four were discordant for both linked markers. Since only concordant sib pairs would have inherited the same growth hormone alleles, further studies to identify mutations of the growth hormone genes should be limited to this subgroup. It is unlikely that the discordance observed in four of the sib pairs is due to recombination, because the polymorphic HincII site is only 116 base-pairs from the -26 codon of the growth hormone gene. Thus, in at least four of the nine families, the mutation responsible for isolated growth hormone deficiency is not within or near the structural gene for growth hormone on chromosome 17.
Complete androgen insensitivity syndrome (CAIS), or so-called testicular feminization, results from the lack of androgen action on target organs. Within this syndrome, two major variants have been described. In the first variant, the specific intracellular androgen receptors are undetectable (CAIS, AR-), whereas normal levels of androgen receptors are measured in the second variant (CAIS, AR+). From studies with cultured labial skin fibroblasts of three CAIS, AR+ patients from the same family, we have demonstrated that their androgen receptors present qualitative differences when compared to normal receptors: 1) the apparent affinity constant (Kd) of 5 alpha-dihydrotestosterone for the receptor is higher than normal; 2) the in vitro dissociation rate of the receptor-steroid complex is faster than normal: 3) the cellular androgen receptor is more thermolabile than normal when cells are exposed to superphysiological temperatures; and 4) the relative binding affinity of the androgen receptor for progesterone is greater than normal. These findings suggest the presence of a structural abnormality of the androgen receptor in patients with CAIS, AR+. However, these changes (e.g. slightly decreased affinity of the receptor for 5 alpha-dihydrotestosterone) probably do not explain the total lack of androgen action in these patients. Finally, the present data from this family along with those from the literature suggest the presence of heterogeneity as to the cause of the defect in CAIS, AR+.
A variety of mild forms of congenital adrenal hyperplasia (CAH) due to partial 21-hydroxylase deficiency have recently been described. We report two families in whom members presented with CAH with various degrees of enzyme deficiency. In family A, two children had the classical salt-losing CAH. Their male sibling and mother presented a very mild asymptomatic form of CAH, characterized by elevated basal plasma levels of 17-hydroxyprogesterone (17-OHP) and exaggerated responses of progesterone and 17-OHP to ACTH stimulation. The Hormonal profile and HLA types of these two individuals suggested allelic compounds, having one mutant gene for classical CAH and another for a mild form. In family B, the proband presented an attenuated form of CAH, manifested by amenorrhea and hirsutism, elevated basal levels of plasma 17-OHP and androgens, as well as markedly increased ACTH response. Two of her four siblings had the same ad HLA type, elevated basal plasma 17-OHP levels, and increased ACTH response. Their father, their paternal aunt, and their paternal uncle had the ab HLA type and normal basal plasma 17-OHP but markedly increased ACTH response. The haplotypes a, b, and d were considered to be linked to a mutation resulting in mild 21-hydroxylase deficiency, the homozygotes with ab HLA type having a milder form of CAH than the homozygotes with the ad HLA type. The wide spectrum of clinical and hormonal characteristics among homozygotes for the 21-hydroxylase deficiency trait suggests that their is a continuum of degree of enzyme deficiency. Furthermore, it suggests that most nonclassical subjects are allelic compounds for variable degrees of severity in the mutation at the 21-hydroxylase locus. More specifically, the study of families A and B shows that the so-called cryptic and attenuated forms of CAH have the same pathophysiological basis.
We studied four patients with Robinow's syndrome. New findings included the following: (1) normal pubertal virilization with persistence of micropenis; (2) elevated basal serum follicle-stimulating hormone levels and a hyperresponse of serum luteinizing hormone to gonadorelin hydrochloride (Factrel) stimulation among postpubertal male patients, suggesting partial primary hypogonadism; (3) normal 5 alpha-reductase and androgen receptor activity in genital skin fibroblasts; and (4) normal to borderline adult height.
An 11-year-old patient with male pseudohermaphroditism presented with essentially normal-appearing female external genitalia. When examined, inguinal gonads, redundant foreskin, and some posterior labial fusion were found. Evaluation revealed basal testosterone (T) levels ranging from 65 to 107 ng/dl with slightly elevated serum gonadotropin levels (luteinizing hormone [LH]: 76 ng/ml, and follicle-stimulating hormone [FSH]: 568 ng/ml). Neither T nor its precursors increased with human chorionic gonadotropin (hCG) stimulation. However, progesterone (P), 17-hydroxyprogesterone (17-OHP), and cortisol (F) responses to adrenocorticotropic hormone (ACTH) were normal. Androgen binding and 5 alpha-reductase activity in cultured genital skin fibroblasts were normal. These data, plus the microscopic finding of a markedly reduced number of Leydig cells, strongly suggest that the male pseudohermaphroditism in this patient was due to inadequate Leydig cell function unrelated to LH receptors.
A patient with 46,XY karyotype and 17 alpha-hydroxylase deficiency is reported who illustrates marked virilization of the external genitalia. Marked phallic development and almost complete labioscrotal fusion with no development of a utriculovaginal pouch were noted. Because the perineum was essentially similar to that seen in male-to-female transsexuals, vaginal construction required the McIndoe procedure. Hence, although some patients with 17 alpha-hydroxylase deficiency have minimal virilization of the external genitalia, this patient's history indicates the operative management necessary in the opposite extreme, when patients show complete masculinization. Hormonal evaluation after gonadectomy revealed the enzyme deficiency based on abnormalities of steroid secretion by the adrenal cortex.
Concentrations of testosterone, androstenedione, dehydroepiandrosterone (DHA), DHA sulfate (DHAS), progesterone, 17 alpha-hydroxyprogesterone (17-OHP), and hydrocortisone were determined in amniotic fluid obtained at amniocentesis or at elective cesarean section. Male fetuses had significantly higher concentrations of testosterone and androstenedione than female fetuses had between 15 and 21 weeks of gestation but not near term (36 to 40 weeks). In both sexes, progesterone and 17-OHP concentrations fell and DHA, DHAS, and hydrocortisone concentrations increased significantly with advancing gestational age. Amniotic fluid 17-OHP, testosterone, DHA, and androstenedione levels from female fetuses with congenital adrenal hyperplasia (CAH) were more elevated in the second trimester than in the third. Three female fetuses at risk for CAH, but not affected, had normal steroid concentrations. Steroid concentrations from two fetuses with Klinefelter's syndrome were not abnormal.
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Eleven cases of true hermaphroditism (eight previously reported) studied at The Johns Hopkins Hospital are presented. The two most recently observed patients had what is to be considered an appropriate endocrine evaluation. This includes karyotyping, and measurement of 24-hour urinary 17-ketosteroids, gonadotropins, plasma testosterone, dihydrotestosterone, and all their precursors before and following a human chorionic gonadotropin stimulation test, and a sexual skin biopsy for androgen receptor binding and 5 alpha-reductase activity. The differential diagnosis of true hermaphroditism is discussed and an approach to gender assignment is presented.
Human skin may be considered as a target organ for androgens, as are male sex accessory organs, since all events involved in testosterone action have been observed in this tissue. As a corollary, the mechanism of androgen action can be studied in vitro in cultured skin fibroblasts. The advantages of this system are that studies can be performed with intact human cells under carefully controlled conditions, differentiated genetic and biochemical characteristics of the cells are faithfully preserved and the biological material is renewable from a single biopsy specimen. The metabolism of androgens, in particular the 5 alpha-reduction of testosterone to the active metabolite, dihydrotestosterone, the intracellular binding of androgen to its specific receptor protein and its subsequent translocation to the nucleus have been studied in skin fibroblasts. The intracellular androgen receptor content of genital skin fibroblasts is higher than that from nongenital skin sites. In addition, the androgen receptor has been characterized as a specific macromolecule with properties of high affinity and low capacity similar to that of other steroid hormone receptors. The pathophysiology of three genetic mutations which alter normal male sexual development and differentiation has been identified in the human skin fibroblast system. In 5 alpha-reductase deficiency, an autosomal recessive disorder in which dihydrotestosterone formation is impaired, virilization of the Wolffian ducts is normal but the external genitalia and urogenital sinus derivatives are female in character. At least two types of X-linked disorders of the androgen receptor exist such that the actions of both testosterone and dihydrotestosterone are impaired and developmental abnormalities may involve both Wolffian derivatives and the external genitalia as well. These two forms of androgen insensitivity result from either the absence of androgen receptor binding activity (receptor (-) form) or apparently normal androgen receptor binding with absence of an appropriate biological response (receptor (+) form). In addition, studies with human skin fibroblasts may also be of value in defining the cellular mechanisms underlying the broad spectrum of partial defects in virilization. In summary, we have correlated our studies of the molecular mechanism of androgen action in human genital skin fibroblasts with those of other investigators as these studies contribute to our understanding of male sexual development and differentiation.
The diagnosis of congenital adrenal hyperplasia due to a deficiency of the enzyme 17 alpha-hydroxylase was made in a genetic male and female sibling pair born of parents who were first cousins. The genetic male was a phenotypic female who presented with primary amenorrhea and mild hypertension. The genetic female exhibited absence of secondary sexual characteristics and severe hypertension. The plasma steroid data confirmed the diagnosis of 17 alpha-hydroxylase deficiency in both subjects: low 17 alpha-hydroxyprogesterone, elevated desoxycorticosterone, elevated corticosterone and elevated progesterone. These are the first case reports of 17 alpha-hydroxylase deficiency in a male-female sibling pair, and they add support to the hypothesis that this is an autosomal recessive disorder.
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Close linkage between HLA and the gene for 21-OH deficiency causing congenital virilizing adrenal hyperplasia (CVAH) has been well documented. HLA-A/B and HLA/GLO recombination data placed the CVAH gene within the HLA-A to GLO interval, with CVAH invariably segregating with HLA. HLA-A,B,C,DR and GLO typing and ACTH stimulation to determine carrier status was done on seven families. Carrier status correlated with the appropriate HLA haplotypes in all offspring, with two exceptions. Two intra-HLA recombinants were detected in one three-generation family. The father of the proband is homozygous for HLA-A2,Cw2,B27 but is a DR2/DR4 heterozygote. The CVAH gene segregated with DR2 in all but one of his offspring who is a carrier and is DR4. This finding is consistent with recombination between the CVAH gene and DR. Study of the father's family confirmed synteny of the CVAH gene and DR2. Three of four sibs of the father inherited this haplotype and were CVAH carrier, as was the paternal grandmother, whose other haplotype was A1, Bw44,DR1. One of the father's sibs was shown serologically to be a HLA-B/D maternal recombinant and a noncarrier. she inherited the A1,Bw44 of one maternal haplotype, but the DR2 of the other. In both recombinants in this family the CVAH gene segregated with the A to B interval, separate from D. While we cannot determine whether the CVAH gene is in the HLA-A to B or B to D interval, this is the first report of two intra-HLA recombinations in one family that unequivocally map the CVAH gene inside HLA-D.