Replacement of arginine 773 by cysteine or histidine in the human androgen receptor causes complete androgen insensitivity with different receptor phenotypes

We have discovered two different point mutations in a single codon of the X-linked androgen-receptor (AR) gene in two pairs of unrelated families who have complete androgen insensitivity (resistance) associated with different AR phenotypes in their genital skin fibroblasts. One mutation is a C-to-T transition at a CpG sequence near the 5' terminus of exon 6; it changes the sense of codon 773 from arginine to cysteine, ablates specific androgen-binding activity at 37 degrees C, and eliminates a unique KpnI site at the intron-exon boundary. The other mutation is a G-to-A transition that changes amino acid 773 to histidine and eliminates an SphI site. This mutant AR has a normal androgen-binding capacity at 37 degrees C but has a reduced affinity for androgens and is thermolabile in their presence. Transient transfection of COS cells with cDNA expression vectors yielded little androgen-binding activity at 37 degrees C from Arg773Cys and abundant activity with abnormal properties from Arg773His, thereby providing the pathogenicity of both sequence alterations. This conclusion coincides with the following facts about evolutionary preservation of the position homologous to Arg773 in the AR: it is occupied by Arg or lysine in the progesterone, glucocorticoid, and mineralocorticoid receptors, and it is within a 14-amino-acid region of their steroid-binding domains that share approximately 85% amino acid identity.     

An exonic point mutation of the androgen receptor gene in a family with complete androgen insensitivity

We have discovered in the X-linked androgen receptor gene a single exonic nucleotide substitution that causes complete androgen insensitivity (resistance) in a sibship with three affected individuals. The mutation, a guanine-to-adenine transition, occurs at nucleotide number 2682 and changes the sense of codon 717 from tryptophan to a translation stop signal. Codon 717 is in exon 4, so the mutation predicts the synthesis of a truncated receptor that lacks most of its androgen-binding domain. The substitution abolishes a recognition sequence for the restriction endonuclease HaeIII. Amplification of exon 4 by the polymerase chain reaction followed by double digestion with HinfI and HaeIII permits facile recognition of hemizygotes and heterozygous carriers of the mutation.     

Androgen receptors and partial androgen insensitivity in male pseudohermaphroditism

Male pseudohermaphroditism can occur either as a result of inadequate fetal androgen secretion or ineffective androgen action. Partial androgen insensitivity syndrome is related to a decrease in the binding capacity of target cells androgen receptors. However, the biochemical expression of these syndromes is variable: although a low concentration of androgen receptors is observed in 75% of the cases, a normal binding capacity is sometimes reported. If the presence of androgen receptors is necessary for hormonal action, it is not sufficient since the receptor has to conserve its functional integrity. Several qualitative abnormalities of the androgen receptor have been described and could thus explain the disturbance in the androgen mechanism of action. The study of androgen-dependent proteins and other androgen-receptor "markers" should offer the clinician a better management of partial androgen insensitivity and allow, in amniotic or trophoblastic cells, a prenatal diagnosis of androgen resistance in high-risk families.     

Genital fibroblasts from normal individuals and patients with androgen insensitivity: demonstration of reduced or absent levels of a specific protein (Mr approximately 41K, pI approximately 6) in receptor negative cells

Genital fibroblasts were obtained from normal individuals and from patients with a variety of syndromes of defective androgenization (complete androgen insensitivity, partial androgen insensitivity, microgenitalia, hypospadias, infertility). Cells were labelled with [35S]methionine, and patterns of protein synthesis compared by two-dimensional gel electrophoresis, with isoelectrofocusing electrophoresis gels or non-equilibrated pH gradient electrophoresis (NEPHGE) gels as the first dimension. A protein (mol. wt approximately 41K, pI approximately 6) was found on NEPHGE gels to be reduced or absent in fibroblasts in which androgen receptor levels were abnormal. The protein was unaltered by prior incubation with 1-100 nM dihydrotestosterone for 48 h, and was present in cells both from normal controls, and from patients with abnormal sexual differentiation showing normal androgen receptor levels. The coincidence of low or absent 41K with low or absent androgen receptors suggested the possibility that it may constitute a steroid-binding moiety of the androgen receptor. To test this possibility cytosols from normal foreskins or normal cultured fibroblasts were adsorbed with testosterone-sepharose affinity resin to remove androgen receptors. Cytosols so treated showed levels of 41K on NEPHGE indistinguishable from those in untreated cytosols, or in cytosols treated with underivatized sepharose. We therefore conclude that the 41K protein, while an accurate marker of the presence or absence of androgen receptors over a range of clinical disorders, is neither an androgen-induced protein nor an androgen-binding protein.     

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.     

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.     

Male pseudohermaphroditism: diagnosis in cell culture.

Testicular feminization is a classic form of complete male pseudohermaphroditism. The individuals have a normal XY karyotype but unambiguously female external genitalia. They have congenital complete insensitivity to androgen due to an X-linked mutation. In four patients (from tow families with several affected members) with the typical phenotype of testicular feminization, a severe deficit of specific androgen-binding activity was detected in cultured fibroblasts from labium majus skin. Measurement of this activity in genital skin fibroblasts improves the differential diagnosis in patients with complete or imcomplete male pseudohermaphroditism before puberty.     

Role of the androgen receptor in male sexual differentiation.

The two androgens responsible for all aspects of male sexual differentiation are testosterone and dihydrotestosterone. The action of both these steroids is mediated by a specific intracellular receptor, the androgen receptor, which is a member of the nuclear receptor superfamily. The androgen receptor gene has been cloned and is located on the X chromosome at Xq11-12. Mutations of this gene have been found in subjects with both complete and partial androgen insensitivity. In a study of 27 subjects with the androgen insensitivity syndrome, we have identified mutations in 14, using a rapid mutation screening assay. The same technique has also been used to determine carrier status in an affected family. We have also identified a mutation in two brothers who show perineal hypospadias as the only evidence of undervirilisation. Familial severe hypospadias should therefore be included as part of the phenotypic spectrum of partial androgen insensitivity. The study of naturally occurring mutations of the androgen receptor gene is providing further information on the function of the androgen receptor and its role in normal male sexual differentiation.     

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.     

Dissecting phenotypic variation among AIS patients

We have created genital skin fibroblast cell lines directly from three patients in a Chinese family affected by androgen insensitivity syndrome (AIS). All patients in the family share an identical AR Arg840Cys mutant but show different disease phenotypes. By using the cell lines, we find that the mutation has not influenced a normal androgen-binding capacity at 37 degrees C but has reduced the affinity for androgens and may cause thermolability of the androgen-receptor complex. The impaired nuclear trafficking of the androgen receptor in the cell lines is highly correlated with the severity of donors' disease phenotype. The transactivity of the mutant is substantially weakened and the extent of the reduced transactivity reflects severity of the donors' disease symptom. Our data reveal that although etiology of AIS is monogenic and the mutant may alter the major biological functions of its wild allele, the function of the mutant AR can also be influenced by the different genetic backgrounds and thus explains the divergent disease phenotypes.     

Prenatal prediction of androgen insensitivity syndrome using exon 1 polymorphism of the androgen receptor gene.

Exon 1 polymorphism of the androgen receptor (AR) gene is characterized by a (CAG)n(CAA) repeat at position 172 following the translation start codon. The aim of this study was to determine whether AR gene exon 1 polymorphism could be used to perform prenatal diagnosis in high risk families with complete or partial androgen insensitivity syndrome. After enzymatic amplification of a 1 kilobase exon 1 fragment, each DNA was simultaneously digested by MspI and PstI restriction enzymes. After electrophoresis on a 15% electrophoresis on a 15% acrylamide gel or a 6% Nusieve gel, we measured the size of the obtained fragments and determined the number of CAG repeats since a 282 basepair fragment corresponds to 21 CAG. We previously showed that the number of CAG repeats within the AR gene exon 1 in 23 families with complete or partial androgen insensitivity syndrome was 19 +/- 4. By this method, we detected heterozygosity in 50% of the mothers. We present here 2 exclusion prenatal diagnoses using exon 1 polymorphism of the AR gene. Family A presented a boy with a severe form of partial androgen insensitivity syndrome. The mother had 2 uncles with ambiguous genitalia. In family B, the affected child had a complete androgen insensitivity syndrome. In both families, analysis of the AR gene exon 1 polymorphism of the trophoblastic DNA showed the presence of the normal maternal X chromosome. The parents decided to carry on the gestation. In family A, the newborn had normal male external genitalia. In family B, sonography confirmed the presence of normal male external genitalia. These data suggest that exon 1 polymorphism of the AR gene could be prenatally used to predict androgen insensitivity syndrome.