Androgen insensitivity in forty-nine patients: classification based on clinical and androgen receptor phenotypes

Androgen receptor binding was studied in genital skin fibroblasts from 49 patients with androgen insensitivity syndrome (AIS) classified as complete (CAIS) or partial (PAIS) based on the clinical phenotype. The majority (64%) of CAIS and a minority (7%) of PAIS patients were receptor negative. Only 3 receptor-positive AIS cell strains of 30 studied failed to show an increase in specific receptor binding after prolonged androgen exposure in vitro. The gene coding for the androgen receptor in such patients appears intact.     

A large deletion/insertion-induced frameshift mutation of the androgen receptor gene in a family with a familial complete androgen insensitivity syndrome

Androgen insensitivity syndrome (AIS) is an X-linked recessive genetic disorder with a normal 46, XY karyotype caused by abnormality of the androgen receptor (AR) gene. One Chinese family consisting of the proband and 5 other members with complete androgen insensitivity syndrome (CAIS) was investigated. Mutation analysis by DNA sequencing on all 8 exons and flanking intron regions of the AR gene revealed a unique large deletion/insertion mutation in the family. A 287 bp deletion and 77 bp insertion (c.933_1219delins77) mutation at codon 312 resulted in a frameshift which caused a premature stop (p.Phe312Aspfs*7) of polypeptide formation. The proband's mother and grandmother were heterozygous for the mutant allele. The proband's father, uncle and grandfather have the normal allele. From the pedigree constructed from mutational analysis of the family, it is revealed that the probably pathogenic mutation comes from the maternal side.     

Molecular analysis of the androgen receptor gene in 52 patients with complete or partial androgen insensitivity syndrome: a collaborative study.

In patients with androgen insensitivity syndrome (AIS), RFLP study of the androgen receptor gene made it possible to analyze whether deletions or mutations could be responsible for abnormalities in androgen responsiveness. We studied RFLPs of DNA from 25 46,XY patients with partial AIS (PAIS), defined as a concentration of androgen receptor in genital-skin fibroblasts less than 340 fmol/mg DNA, and DNA from 27 46,XY patients with complete AIS (CAIS) with no detectable androgen receptor site. DNA samples were digested with BamHI, EcoRI, HindIII and TaqI restriction enzymes and hybridized with three cDNA probes covering the three domains of the androgen receptor. When we had the maternal and an unaffected brother's DNA, we analyzed the two androgen receptor gene polymorphisms described, the HindIII and the exon 1 CAG repeat polymorphisms, in order to distinguish the two maternal X chromosomes, and to detect carriers of AIS. We did not find any large deletion among the 52 patients. We observed a heterozygous mother in 3 of 14 families studied with the HindIII polymorphism, and in 12 of 25 families using the exon 1 CAG repeat polymorphism. This study suggests that in AIS, abnormalities in androgen receptor response could be related to point mutations or microdeletions rather than to gross structural alterations of the androgen receptor gene. Furthermore, unless the point mutation has been described, exon 1 and HindIII polymorphism studies would enable the identification of carriers in 50% of families, and the prenatal diagnosis of AIS.     

L712V mutation in the androgen receptor gene causes complete androgen insensitivity syndrome due to severe loss of androgen function

Inability to respond to the circulating androgens is named as androgen insensitivity syndrome (AIS). Mutations in the androgen receptor (AR) gene are the most common cause of AIS. A cause and effect relationship between some of these mutations and the AIS phenotype has been proven by in vitro studies. Several other mutations have been identified, but need to be functionally validated for pathogenicity. Screening of the AR mutations upon presumptive diagnosis of AIS is recommended. We analyzed a case of complete androgen insensitivity syndrome (CAIS) for mutations in the AR gene. Sequencing of the entire coding region revealed C > G mutation (CTT–GTT) at codon 712 (position according to the NCBI database) in exon 4 of the gene, resulting in replacement of leucine with valine in the ligand-binding domain of the AR protein. No incidence of this mutation was observed in 230 normal male individuals analyzed for comparison. In vitro androgen binding and transactivation assays using mutant clone showed approximately 71% loss of ligand binding and about 76% loss of transactivation function. We conclude that CAIS in this individual was due to L712V substitution in the androgen receptor protein.     

Transactivation properties of wild-type and mutant androgen receptors in transiently transfected primary human fibroblasts

BACKGROUND: Stromal cells play key roles during androgen-mediated male sexual differentiation. Our objective was to establish a transient transfection method for primary human fibroblasts enabling functional characterization of wild-type (wt) and mutant androgen receptor (AR) plasmid constructs, corresponding to partial and complete androgen insensitivity syndrome (PAIS/CAIS). METHODS: An AR-negative fibroblast strain (ARD842) was established from the gonads of a CAIS patient. Wt-AR or either mutants L712F (PAIS), R774C or V866M (CAIS) were transfected using a polyamine-based procedure. Alternatively, two AR-positive male foreskin fibroblast strains were investigated. Androgen-induced activation of two co-transfected reporter plasmids ((ARE)(2)TATA-, MMTV-luciferase) was measured. RESULTS: All three fibroblast strains showed a ligand-dependent rise of luciferase activity after transfection of wt-AR. Mutant plasmids were assessed in AR-negative ARD842 cells. While L712F showed high partial activity, R774C and V866M were nearly inactive. The intrinsic AR of normal foreskin fibroblasts revealed no measurable ligand-inducible reporter gene activity. CONCLUSIONS: Polyamine-based transfection of AR plasmids into cultured fibroblasts provides a promising tool for analysis of AR transactivation, thereby considering a stromal cellular background. This is supported by the mutant ARs which showed the expected levels of impaired transactivation with respect to the corresponding AIS phenotypes. The role of the intrinsic AR in normal male human foreskin fibroblasts needs further exploration.     

Clinical and genetic characterization of complete androgen insensitivity syndrome in a Chinese family

We studied a family with two cousins who were diagnosed with complete androgen insensitivity syndrome, an X-linked disorder caused by mutations in the androgen receptor gene. A pedigree analysis and a molecular study using PCR and DNA sequencing clarified each female family member's androgen receptor status and revealed a mutation consisting of the deletion of exon 2 and surrounding introns of the androgen receptor gene. Based on the relative nucleotide positions, we concluded that the deletion mutation in exon 2 and its surrounding introns was approximately 6000 to 7000 bp. This mutation, never previously fully characterized using DNA sequencing, was responsible for complete androgen insensitivity syndrome in this family. Pedigree analysis with a molecular study of the androgen receptor gene in affected families facilitates genetic counseling provided to family members.     

Point mutation in the steroid-binding domain of the androgen receptor gene in a family with complete androgen insensitivity syndrome (CAIS)

An exonic single nucleotide substitution in the human androgen receptor gene (hAR) could be detected in an Italian family with two children affected by complete androgen insensitivity syndrome (CAIS), also called testicular feminization. This mutation leads to a guanine to adenine transition in exon 5, changing the sense of the codon from methionine (ATG) to valine (GTG). As this mutation abolishes a NcoI restriction site, a rapid test for the mutation can be performed by digestion of the polymerase chain reaction products with this enzyme. Previous results of indirect gene diagnosis in this family could be confirmed by this method.     

Association of the Hind III polymorphism with the androgen receptor gene in partial androgen insensitivity syndrome.

Partial androgen insensitivity syndrome (PAIS) is an X-linked disorder resulting from defects in the intracellular androgen receptor (AR). The cloning of the AR cDNA has provided the molecular tools to identify gene abnormalities. Gene deletions being the exception in PAIS, prenatal diagnosis of PAIS resulting from a single base mutation in high risk families is not practical unless the mutation is already known. Brown et al. (1989) reported that 10% of normal X chromosomes present a Hind III 6.7/3.5 kb polymorphism. In this study, we report the association of the Hind II polymorphism in a woman whose son has a PAIS associated with a very low androgen receptor concentration: we differentiated the two maternal X chromosomes and characterized the affected allele. These data demonstrate that the presence of Hind III polymorphic fragments could be used in prenatal diagnosis of androgen insensitivity syndrome in high risk families.     

Phenotypic heterogeneity associated with identical mutations in residue 870 of the androgen receptor

BACKGROUND/AIMS: Mutations in the androgen receptor (AR) gene result in an X-linked recessive form of male pseudohermaphroditism known as the androgen-insensitivity syndrome (AIS). The alterations most frequently observed are missense or nonsense point mutations in exons 4-8 of the AR gene that affect the steroid-binding domain of the receptor in subjects with various degrees of androgen resistance. Despite the increasing number of AR mutations identified, a reliable genotype-phenotype correlation has not been established and individuals with the same molecular defect may exhibit different phenotypes. Here, we studied a patients with an AIS characterized by bilateral gynecomastia, normal male external genitalia, and normal sperm counts. METHODS: Exon-specific polymerase chain reaction, single-stranded conformational polymorphism, and sequencing analysis of the subject's AR gene were performed in addition to hormone-binding assays in skin fibroblasts from the patient. RESULTS: A point mutation at codon 870 of the AR, changing alanine to valine, was detected. CONCLUSION: As AR missense mutations changing alanine 870 to valine have been previously described in 3 unrelated patients showing severe AIS phenotypes, we conclude that phenotypic heterogeneity associated to identical mutations in the AR gene is probably due to individual functional differences in AR coregulator molecules.     

Photoaffinity labelling with [3H]R1881 of androgen receptors from human cultured genital fibroblasts of normal individuals and patients with androgen receptor abnormalities

Androgen receptors were partially purified by affinity chromatography of cytosols prepared from cultured genital fibroblasts of normal individuals and patients with androgen receptor abnormalities. Partially purified receptors were covalently labelled with [3H]R1881 (triated methyltrienolone) by ultraviolet photoactivation. Gel electrophoresis of cytosols from normal individuals showed a single radioactive peak, Mr approximately 97 K. Cytosols from patients with decreased nuclear transfer showed a similar peak, Mr approximately 97 K; cytosols from patients with partial androgen insensitivity (PAIS) or receptor positive complete androgen insensitivity (CAIS, R+) showed a peak Mr approximately 30 K-43 K.     

The human androgen receptor: Structure/function relationship in normal and pathological situations

Discrete functions have been attributed to precise regions of the human androgen receptor (hAR) by expression of deletion mutants in COS and HeLa cells. A large C-terminal domain constitutes the hormone-binding region and a central basis, cysteine-rich domain is responsible for DNA binding. In addition, separate domains responsible for transactivation and nuclear translocation have been identified. In LNCaP cells (a prostate tumor cell line) the hAR is a heterogeneous protein which is synthesized as a single 110 kDa protein, but becomes rapidly phosphorylated to a 112 kDa protein. Metabolic labeling experiments using radioactive orthophosphate also indicated that the hAR is a phosphoprotein. Structural analysis of the AR gene in LNCaP cells and in 46, XY-individuals displaying androgen insensitivity (AIS) has revealed several different point mutations. In LNCaP cells the mutation affects both binding specificity and transactivation by different steroids. In a person with complete AIS a point mutation was identified in the splice donor site of intron 4, which prevents normal splicing and activates a cryptic splice donor site in exon 4. The consequence is a functionally inactive AR protein due to an in-frame deletion in the steroid-binding domain. In two unrelated individuals with complete AIS, two different single nucleotide alterations in codon 686 (Asp) were found. Both mutations resulted in functionally inactive ARs due to rapidly dissociating hormone-AR complexes. It is concluded that the hAR is a heterogeneous phosphoprotein in which functional errors have a dramatic impact on phenotype and fertility of 46, XY-individuals.     

Increased peripheral aromatase activity in prepubertal children with partial androgen insensitivity syndrome

It has been suggested that rate of estrogen formation was higher in patients with androgen insensitivity syndrome (AIS). This work was designed to find out if peripheral aromatase activity could be related to a defect in androgen action in prepubertal children with male pseudohermaphroditism. Fibroblast estrogen production was assayed by a highly specific enzymatic determination. Foreskin fibroblast strains were raised from 17 children with partial androgen insensitivity (PAIS) as defined by dihydrotestosterone binding activity in cells. Results are expressed as pmol estrogens/mg proteins synthetized/day when cultured fibroblasts are incubated with D4-androstenedione. In normal prepubertal boys (n = 19), aromatase activity ranged between 5 and 10 pmol estrogens/mg proteins/day, while in postpubertal boys it varied between 15 and 34 pmol estrogens/mg proteins/day. In prepubertal boys with PAIS (n = 17) aromatase activity is highly elevated: 19.4 +/- 8.4 pmol/mg proteins/day. These results show that (a) peripheral aromatase activity is low before puberty and (b) fibroblast estrogen synthesis is significantly (p less than 0.001) increased in prepubertal children with PAIS. Our data suggest that low utilization of androgens by target cells stimulates the production of estrogen. Peripheral aromatase activity can thus be considered as a 'marker' of androgen insensitivity in prepubertal children with male pseudohermaphroditism.     

Syndromes d’insensibilite aux androgenes: Aspects moleculaires

The molecular analysis of clinical syndromes involving androgen insensitivity has been facilitated by the availability of increasingly powerful molecular biological techniques. Complementary DNA of the androgen receptor gene (wich was recently cloned and sequenced) has been used as a probe to investigate DNA restriction fragment length polymorphisms in patients with partial or complete androgen insensitivity. Such studies have demonstrated that deletions are rare. Using enzymatic amplification and sequencing of exons of the androgen receptor gene, several groups have described point mutations in patients with androgen insensitivity. PCR, accompanied by denaturing gradient gel electrophoresis or single strand conformation polymorphism assays, has revealed further mutations. These powerful tools, together with studies of mRNA from expression of the mutant gene, have also illustrated structure-function associations of the androgen receptor gene in some patients with androgen insensitivity.