Complete androgen insensitivity syndrome caused by a novel mutation in the ligand-binding domain of the androgen receptor: functional characterization

Mutations in the X-linked androgen receptor (AR) gene cause the androgen insensitivity syndrome by impairing androgen-dependent male sexual differentiation to varying degrees. Complete androgen insensitivity (CAIS) yields an external female phenotype, whereas affected cases of partial androgen insensitivity have various ambiguities of the genitalia. Here we describe a 46,XY phenotypically female patient with all of the characteristics of CAIS, i.e. primary amenorrhea, no axillary or pubic hair, female external genitalia, no uterus, and undescended testes. Defects in testosterone and dihydrotestosterone synthesis were excluded. The molecular basis of the disease was clarified by means of direct sequencing of PCR-amplified exonic fragments of the AR gene. An A to C transition in exon 4 of the AR gene led to a novel missense His(689)Pro mutation in the ligand-binding domain of the AR protein. Functional studies demonstrated that the mutated AR is unable to efficiently bind its natural ligand dihydrotestosterone and to trans-activate known androgen response elements. Analysis of the structural consequences of the His(689)Pro substitution suggests that this mutation is likely to perturb the conformation of the second helix of the AR ligand-binding domain, which contains residues critical for androgen binding.     

雄激素受体基因新突变致雄激素不敏感综合征

目的 分析2例雄激素不敏感综合征患者及其家系的临床及分子遗传学.方法 收集2例雄激素小敏感综合征患者的临床资料,从患者及其家系成员的外周血单个核细胞抽提基因组DNA,应用PCR扩增雄激素受体基因并直接测序,明确患者及其父母基因有无突变.结果 患者1表现为女性外生殖器、单侧乳房发育、原发性闭经、阴毛腋毛缺如.患者2表现为男性化不全,体毛稀少、双侧乳房发育、尿道下裂.基因检测证实患者1雄激素受体基因第2号外显子第579位密码子点突变(S579N),并证实为一新突变.患者2第5号外显子第747位密码子点突变(V747M).结论 该2例雄激素受体不敏感综合征系分别由雄激素受体基因S579N及V747M所致,其中S579N突变尚未见文献报道.     

A novel mutation (N233K) in the transactivating domain and the N756S mutation in the ligand binding domain of the androgen receptor gene are associated with male infertility*

OBJECTIVE: Resistance to androgens has been suggested as a possible cause of male infertility. This hypothesis is based mainly on binding studies in genital skin fibroblasts but the molecular evidence is sparse. DESIGN: Molecular studies of the androgen receptor gene were performed in 10 azoo- or oligozoospermic men, presenting with clinical signs of low androgen activity-poor virilization and high serum LH despite elevated testosterone levels, but without genital malformations. PATIENTS: Ten men with serum LH >10 IU/l and testosterone >30 nmol/l as well as a low sperm concentration < 20 x 106/ml. MEASUREMENTS: Genomic DNA was prepared from peripheral leucocytes and PCR-amplification of the coding region of androgen receptor was performed, followed by direct sequencing. Identified mutations were reconstructed by site-directed mutagenesis and the functional properties of the mutants were analysed, using transient expression in COS-1 cells and subsequent transactivation assays. Hormone binding assays were performed in genital skin fibroblasts from the patients. RESULTS: Two of the 10 men were shown to have a mutation in the androgen receptor gene. Subject 1, who presented with azoospermia, serum testosterone (T) 50 nmol/l and LH 20 IU/l, had a mutation in exon 1, changing amino acid asparagine 233 to lysine (N233K). In fibroblasts cultured from genital skin, the receptor affinity for 5alpha-dihydrotestosterone (DHT) was normal as compared to healthy controls, but the receptor-hormone complex was thermolabile at 42 degrees C. Subject 2 exhibited severe oligozoospermia and a similar endocrine pattern (T = 50 nmol/l and LH = 25 IU/l). He had a mutation in exon 5 changing asparagine 756 to serine (N756S). The affinity for DHT in cultured genital fibroblasts from this patient was reduced. Transactivation was abnormal for both mutants, N233K reaching 46% and N756S 38% of wild type activity when stimulated with 10 nmol/l DHT. CONCLUSIONS: Androgen receptor mutations may affect sperm production without resulting in genital malformations. Thus, in infertile men with a clinical presentation of poor androgen activity and an endocrine profile compatible with androgen resistance, mutations in the androgen receptor should be taken into consideration.     

A novel point mutation in helix 11 of the ligand-binding domain of the human glucocorticoid receptor gene causing generalized glucocorticoid resistance

BACKGROUND: Generalized glucocorticoid resistance is a rare condition characterized by partial, end-organ insensitivity to glucocorticoids, compensatory elevations in adrenocorticotropic hormone and cortisol secretion, and increased production of adrenal steroids with androgenic and/or mineralocorticoid activity. We have identified a new case of glucocorticoid resistance caused by a novel mutation of the human glucocorticoid receptor (hGR) gene and studied the molecular mechanisms through which the mutant receptor impairs glucocorticoid signal transduction. METHODS AND RESULTS: We identified a novel, single, heterozygous nucleotide (T --> C) substitution at position 2209 (exon 9alpha) of the hGR gene, which resulted in phenylalanine (F) to leucine (L) substitution at amino acid position 737 within helix 11 of the ligand-binding domain of the protein. Compared with the wild-type receptor, the mutant receptor hGRalphaF737L demonstrated a significant ligand-exposure time-dependent decrease in its ability to transactivate the glucocorticoid-inducible mouse mammary tumor virus promoter in response to dexamethasone and displayed a 2-fold reduction in the affinity for ligand, a 12-fold delay in nuclear translocation, and an abnormal interaction with the glucocorticoid receptor-interacting protein 1 coactivator. The mutant receptor preserved its ability to bind to DNA and exerted a dominant-negative effect on the wild-type hGRalpha only after a short duration of exposure to the ligand. CONCLUSIONS: The mutant receptor hGRalphaF737L causes generalized glucocorticoid resistance because of decreased affinity for the ligand, marked delay in nuclear translocation, and/or abnormal interaction with the glucocorticoid receptor-interacting protein 1 coactivator. These findings confirm the importance of the C terminus of the ligand-binding domain of the receptor in conferring transactivational activity.     

A mutation of the androgen receptor associated with partial androgen resistance, familial gynecomastia, and fertility

A family is described in which gynecomastia and undervirilization in five men (four of whom have fathered children) were inherited in a manner compatible with an X-linked defect. Three members from whom blood could be obtained had supranormal serum testosterone and normal LH and FSH levels. One man had severe oligospermia with decreased motility, and one had normal sperm density and motility. In fibroblasts cultured from genital skin biopsies from two of the men, the levels of androgen receptor and affinity of binding of receptor to dihydrotestosterone were normal. However, androgen binding in fibroblast monolayers was thermolabile, up-regulation of receptor levels did not occur after prolonged incubation of monolayers with dihydrotestosterone or methyltrienolone, and dissociation rates at 37 C were increased with the synthetic androgen mibolerone. In addition, in cytosol preparations the androgen receptor protein was unstable. This disorder probably represents the most subtle functional abnormality of androgen receptor characterized to date, since it is compatible with normal male phenotypic development and in some affected men with fertility. It follows that infertility is not an invariable feature of androgen resistance as we previously suggested.     

Oligozoospermia with normal fertility in male mice lacking the androgen receptor in testis peritubular myoid cells

Androgens and the androgen receptor (AR) play important roles in the testes. Previously we have shown that male total AR knockout (T-AR-/y) mice revealed incomplete germ cell development and lowered serum testosterone levels, which resulted in azoospermia and infertility. However, the consequences of AR loss in particular types of testicular cells remain unclear. Using a Cre-loxP conditional knockout strategy, we generated a tissue-selective knockout mouse with the AR gene deleted in testis peritubular myoid cells (PM-AR-/y). Phenotype analyses showed that PM-AR-/y mice were indistinguishable from WT AR (AR+/y) mice with the exception of smaller testes size. PM-AR-/y mice have serum testosterone concentrations comparable with AR+/y mice. PM-AR-/y mice have oligozoospermia in the epididymis; however, fertility was normal. Although normal germ cell distribution ratio was found, total germ cell number decreased in PM-AR-/y mice. Further mechanistic studies demonstrated that PM-AR-/y mice have defects in the expression of Sertoli cells' functional marker genes such as tranferrin, epidermal fatty acid-binding protein, androgen-binding protein, and other junction genes including occludin, testin, nectin, zyxin, vinculin, laminingamma3, gelsolin, connection43, and N-cadherin. Furthermore, there were defects in peritubular myoid cell contractility-related genes such as endothelin-1, endothelin receptor A and B, adrenomedullin, adrenomedullin receptor, and vasopressin receptor 1a. Together, our PM-AR-/y mice provide in vivo evidence for the requirement of functional AR in peritubular myoid cells to maintain normal Sertoli cells function and peritubular myoid cell contractility, thus ensuring normal spermatogenesis and sperm output.     

Generalized resistance to thyroid hormone associated with a mutation in the ligand-binding domain of the human thyroid hormone receptor beta.

The syndrome of generalized resistance to thyroid hormone is characterized by elevated circulating levels of thyroid hormone in the presence of an overall eumetabolic state and failure to respond normally to triiodothyronine. We have evaluated a family with inherited generalized resistance to thyroid hormone for abnormalities in the thyroid hormone nuclear receptors. A single guanine----cytosine replacement in the codon for amino acid 340 resulted in a glycine----arginine substitution in the hormone-binding domain of one of two alleles of the patient's thyroid hormone nuclear receptor beta gene. In vitro translation products of this mutant human thyroid hormone nuclear receptor beta gene did not bind triiodothyronine. Thus, generalized resistance to thyroid hormone can result from expression of an abnormal thyroid hormone nuclear receptor molecule.     

Complete androgen insensitivity caused by a new frameshift deletion of two base pairs in exon 1 of the human androgen receptor gene.

We describe a novel mutation in exon 1 of the androgen receptor gene in a patient with complete androgen insensitivity (CAIS). Endocrine findings were typical for androgen insensitivity (testosterone serum levels in the upper limit of normal males and increased LH serum concentrations). Biochemical investigations in cultured genital skin fibroblasts of the patient showed a normal 5alpha-reductase activity but a complete absence of androgen binding. Western blot analysis revealed no detectable protein product. Sequence analysis of the entire coding region of the androgen receptor gene resulted in the identification of a 2-bp deletion in codon 472, causing frameshift and introduction of a premature stop codon 27 codons downstream of the mutation.     

The molecular structure of the Toll-like receptor 3 ligand-binding domain

Innate immunity is the first line of defense against invading pathogens. Toll-like receptors (TLRs) act as sentinels of the innate immune system, sensing a variety of ligands from lipopolysaccharide to flagellin to dsRNA through their ligand-binding domain that is composed of leucine-rich repeats (LRRs). Ligand binding initiates a signaling cascade that leads to the up-regulation of inflammation mediators. In this study, we have expressed and crystallized the ectodomain (ECD) of human TLR3, which recognizes dsRNA, a molecular signature of viruses, and have determined the molecular structure to 2.4-A resolution. The overall horseshoe-shaped structure of the TLR3-ECD is formed by 23 repeating LRRs that are capped at each end by specialized non-LRR domains. The extensive beta-sheet on the molecule's concave surface forms a platform for several modifications, including insertions in the LRRs and 11 N-linked glycans. The TLR3-ECD structure indicates how LRR loops can establish distinct pathogen recognition receptors.     

A familial case of congenital hypothyroidism caused by a homozygous mutation of the thyrotropin receptor gene.

Most of the time congenital hypothyroidism appears as a sporadic disease. In addition to the rare defects in hormonosynthesis associated with goiters, the causes of congenital hypothyroidism include agenesis and ectopy of the thyroid gland. The study of some familial cases has allowed the identification of a few genes responsible for congenital hypothyroidism. We report here a familial case of congenital hypothyroidism, transmitted as a recessive trait, and caused by a homozygous mutation in the thyrotropin receptor (TSH-R). The initial diagnosis of thyroid agenesis, based on the absence of tracer uptake on scintiscan, was incorrect, because ultrasound examination identified severely hypoplastic thyroid tissue in the cervical region.     

Novel missense mutation in the P-box of androgen receptor in a patient with androgen insensitivity syndrome

Mutations in the X-linked AR gene cause androgen insensitivity syndrome (AIS) by impairing androgen-dependent male sex differentiation to various degree. Here we describe a partial AIS patient with confliction with the assigned female sex. Although the patient was noticed to have ambiguous genitalia at birth, the patient was reared as a female with no medical intervention. At the age of 31 years, the patient visited us because the patient was dissatisfied with the assigned female sex. The patient was treated with systemic testosterone and topical dihydrotestosterone, but the external genitalia responded only minimally to the treatment. The genetic analysis revealed a novel missense K580R mutation in the P-box of the DNA-binding domain of androgen receptor, which was the first missense mutation shared by AIS and prostate cancer. Although the best predictor of the adult gender identity is documented to be the initial gender assignment in patients with partial AIS as well as those with complete AIS, deciding gender assignment for infants with partial AIS is still challenging.     

Impact of estrogen replacement therapy in a male with congenital aromatase deficiency caused by a novel mutation in the CYP19 gene

Recent reports of the impact of estrogen receptor alpha and aromatase deficiency have shed new light on the importance of estrogen for bone formation in man. We describe a novel mutation of the CYP19 gene in a 27-yr-old homozygous male of consanguinous parents. A C to A substitution in intron V, at position -3 of the splicing acceptor site before exon VI of the CYP19 gene, is the likely cause of loss of aromatase activity. The mRNA of the patient leads to a frameshift and a premature stop codon 8 nucleotides downstream the end of exon V. Both parents were shown to be heterozygous for the same mutation. Apart from genua valga, kyphoscoliosis, and pectus carniatus, the physical examination was normal including secondary male characteristics with normal testicular size. To substitute for the deficiency, the patient was treated with 50 micro g transdermal estradiol twice weekly for 3 months, followed by 25 micro g twice weekly. After 6 months estrogen levels (<20 at baseline and 45 pg/ml at 6 months; normal range, 10-50) and estrone levels (17 and 34 ng/ml; normal range, 30-85) had normalized. Bone maturation progressed and the initially unfused carpal and phalangeal epiphyses began to close within 3 months and were almost completely closed after 6 months. The bone age, assessed by roentgenographic standards for bone development by Gruelich and Pyle, was 16.5 at baseline and 18-18.5 yr after 6 months of treatment. Bone density of the distal radius (left), assessed by quantitative computed tomography, increased from 52 to 83 mg/cm(3) (normal range, 120-160) and bone mineral density of the lumbar spine, assessed by dual-energy x-ray-absorptiometry, increased from 0.971 to 1.043 g/cm(2) (normal range, >1.150). Osteocalcin as a bone formation parameter increased from 13 to 52 micro g/l (normal range, 24-70) and aminoterminal collagen type I telopeptide as a bone resorption parameter increased from 62.9 to 92.4 nmol/mmol creatinine (normal range, 5-54). Semen analysis revealed oligoazoospermia (17.4 million/ml; normal >20) at baseline. After 3 months of treatment, the sperm count increased (23.1 million/ml) and decreased rapidly (1.1 million/ml) during the following 3 months. The sperm motility was reduced at baseline and decreased further during treatment. Area under the curve of insulin, C-peptide, and blood glucose levels during oral glucose tolerance test decreased after 6 months (insulin: 277 vs. 139 micro U/ml.h; C-peptide 52 vs. 15 ng/m.h; area under the curve glucose: 17316 vs. 12780 mg/d.min). Triglycerides (268 vs. 261 mmol/liter) and total cholesterol levels (176 vs. 198 mmol/liter) did not change significantly, but the low-density lipoprotein/high-density lipoprotein ratio decreased from 5.37 to 3.56 and lipoprotein (a) increased from 19.9 to 60.0 mg/dl (normal range, <30). In this rare incidence of estrogen deficiency, estrogen replacement demonstrated its importance for bone mineralization and maturation and glucose metabolism in a male carrying a novel mutation in the CYP19 gene.     

Polymorphic CAG repeats of the androgen receptor gene in Japanese male patients with ankylosing spondylitis

OBJECTIVE: In view of a possible role of androgens in the pathogenesis of ankylosing spondylitis (AS), we investigated the association between Japanese male patients with AS and CAG microsatellites of the androgen receptor (AR) gene which related to the AR transactivation function. METHODS: Peripheral blood was collected from 39 men with AS and 305 male control subjects. The number of CAG repeats in exon 1 of the AR gene was determined. RESULTS: CAG repeat lengths in AS patients were significantly shorter than those in the controls (median value 22 vs 23; P = 0.03). However, there was no significant difference in CAG repeats between HLA-B27-positive and -negative patients (median value 22 vs 22; P = 0.78). CONCLUSIONS: Shorter CAG repeats of the AR gene, presenting high levels of transactivation activity, may play a role in male AS.     

Novel point mutation in the splice donor site of exon-intron junction 6 of the androgen receptor gene in a patient with partial androgen insensitivity syndrome

Androgen receptor (AR) gene mutations have been shown to cause androgen insensitivity syndrome with altered sexual differentiation in XY individuals, ranging from a partial insensitivity with male phenotype and azoospermia to a complete insensitivity with female phenotype and the absence of pubic and axillary sexual hair after puberty. In this study we present an 11-yr-old XY girl, with clinical manifestations peculiar for impaired androgen biological action, including female phenotype, blind-ending vagina, small degree of posterior labial fusion, and absence of uterus, fallopian tubes, and ovaries. At the time of the diagnosis the patient had a FSH/LH ratio according to the puberal stage, undetectable 17beta-estradiol, and high levels of testosterone (80.1 ng/mL). After bilateral gonadectomy, performed at the age of 11 yr, histological examination showed small embryonic seminiferous tubules containing prevalently Sertoli cells and occasional spermatogonia together with abundant fibrous tissue. Molecular study of the patient showed a guanine to thymine transversion in position +5 of the donor splice site in the junction between exon 6 and intron 6 of the AR gene. The result of RT-PCR amplification of the AR messenger ribonucleic acid from cultured genital skin fibroblasts of the patient suggests that splicing is defective, and intron 6 is retained in most of the receptor messenger ribonucleic acid molecules. We show by immunoblotting that most of the expressed protein lacks part of the C-terminal hormone-binding domain, and a small amount of normal receptor is observed. This is probably responsible for the reduced binding capacity in genital skin fibroblasts of the patient. The molecular basis of the alteration in this case is a novel, uncommon mutation, leading to a phenotype indicative of a partial androgen insensitivity syndrome, Quigley's grade 5.     

Retinoic acid-resistant HL-60R cells harbor a point mutation in the retinoic acid receptor ligand-binding domain that confers dominant negative activity.

Retinoic acid (RA) induces granulocytic differentiation of acute promyelocytic leukemia (APL) cells and is a useful therapeutic agent for patients with this disease. In the HL-60 promyelocytic leukemia cell line, this RA-induced granulocytic differentiation appears to be directly mediated through the RA receptor (RAR-alpha). We have previously identified a mutant subclone of HL-60 (designated HL-60R) that exhibits relative resistance to RA and that harbors RA receptors with markedly reduced affinity for RA. In the present study, we have now identified the genetic basis for this aberrant RA receptor activity. DNA sequencing of polymerase chain reaction-amplified cDNA products corresponding to the RAR-alpha ligand-binding domain shows a point mutation in RAR-alpha codon 411 in this mutant HL-60R subclone. This specific C-->T mutation generates a termination codon resulting in the truncation of 52 amino acids at the COOH terminal end of RAR-alpha. In cotransfection studies, expression vectors harboring this mutated RAR-alpha exhibit dominant negative activity with respect to the trans-activating function of the normal RAR-alpha. Although our observations are limited to HL-60 cells, similar RA receptor mutations might play an important role in the acquisition of RA resistance in RA-treated APL patients.