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.     

Molecular defects of the androgen receptor

Defects of the androgen receptor cause a wide spectrum of abnormalities of phenotypic male development, ranging from individuals with mild defects of virilization to those with complete female phenotypes. In parallel with this phenotypic spectrum, a large number of different mutations have been identified that alter the synthesis or functional activity of the receptor protein. In many instances, the genetic mutations identified lead to an absence of the intact, full-length receptor protein. Such defects (splicing defects, termination codons, partial or complete gene deletions) invariably result in the phenotype of complete androgen insensitivity (complete testicular feminization). By contrast, single amino acid substitutions in the androgen receptor protein can result in the entire phenotypic spectrum of androgen resistant phenotypes and provide far more information on the functional organization of the receptor protein. Amino acid substitutions in different segments of the AR open-reading frame disturb AR function by distinct mechanisms. Substitutions in the DNA binding domain of the receptor appear to comprise a relatively homogeneous group. These substitutions impair the capacity of the receptor to bind to specific DNA sequence elements and to modulate the function of responsive genes. Amino acid substitutions in the hormone-binding domain of the receptor have a more varied effect on receptor function. In some instances, the resulting defect is obvious and causes an inability of the receptor to bind hormone. In other instances, the effect is subtler, and may result in the production of a receptor protein that displays qualitative abnormalities of hormone binding or from which hormone dissociates more rapidly. Often it is not possible to correlate the type of binding defect with the phenotype that is observed. Instead, it is necessary to measure the capacity of the receptor that is synthesized in functional assays in order to discern any type of correlation with phenotype. Finally, two types of androgen receptor mutation do not fit such a categorization. The first of these—the glutamine repeat expansion that is observed in spinal and bulbar muscular atrophy—leads to a reduction of receptor function that can be measured in heterologous cells or in fibroblasts established from such patients. The expression of ARs containing such expanded repeats in men is associated with a degeneration of motor neurons in the spinal cords of affected patients. Likewise, the alterations of androgen receptor structure that have been detected in advanced forms of prostate cancer also behave as gain-of-function mutations. In this latter type of mutation, the exquisite specificity of the normal androgen receptor is relaxed and the mutant receptors can be activated by a variety of steroidal and non-steroidal ligands.     

Androgen resistance due to decreased amounts of androgen receptor: a reinvestigation

To provide insight into the pathogenesis of the androgen resistance in a previously described family with X-linked Reifenstein syndrome, we have systematically assessed a variety of parameters of androgen receptor function in fibroblasts cultured from scrotal skin biopsies. We assessed the amount of high affinity binding, the affinity of ligand binding to the receptor, the upregulation of androgen receptor levels by androgen, the stability of ligand binding in intact fibroblasts at high temperature, the dissociation of ligand from the receptor, the intranuclear localization and salt elution profiles of ligand-receptor complexes, and the ultracentrifugation characteristics of the ligand-receptor complexes. Since the only parameter found to be abnormal is a decreased amount of receptor, we conclude that the underlying mutation in this family influences the amount rather than the structure of the androgen receptor protein.     

Cloning, structure and expression of a cDNA encoding the human androgen receptor

A cDNA clone has been isolated from a library prepared of mRNA of human breast cancer T47D cells with an oligonucleotide probe homologous to part of the region encoding the DNA-binding domain of steroid receptors. The clone has a size of 1505 bp and sequence analysis revealed an open reading frame of 1356 bp. The deduced amino acid sequence displays two highly conserved regions identified as the putative DNA-binding and hormone binding domains respectively of steroid receptors. Expression of this cDNA clone in COS cells produces a nuclear protein with all the binding characteristics of the human androgen receptor (hAR). The gene encoding the cDNA is assigned to the human X-chromosome. High levels of three hybridizing mRNA species of 11, 8.5 and 4.7 kb respectively are found in the human prostate cancer cell line (LNCaP), which contains elevated levels of hAR. The present data provide evidence that we have isolated a cDNA that encodes a major part of the human androgen receptor.     

A unique mutation in the vitamin D receptor gene in three Japanese patients with vitamin D-dependent rickets type II: utility of single-strand conformation polymorphism analysis for heterozygous carrier detection.

Vitamin D-dependent rickets type II is a hereditary disease resulting from a defective vitamin D receptor. In three Japanese patients with vitamin D-dependent rickets type II whose fibroblasts displayed normal cytosol binding and impaired nuclear uptake of 1,25-dihydroxyvitamin D3, western, Southern, and northern analyses failed to disclose any abnormalities in vitamin D3 receptor protein and its gene. Exons 2 and 3 of the vitamin D receptor cDNA, which encode the DNA-binding domain consisting of two zinc fingers, were amplified by PCR and sequenced to identify the specific mutations in the vitamin D receptor gene. In the three patients and one normal control a T-to-C transition was found in the putative initiation codon, while this transition was not observed in another normal control. This finding suggested that an original initiation codon was located at positions 10-12 in the human vitamin D receptor cDNA sequence reported previously. In contrast, a unique G-to-A transition at position 140 in exon 3, resulting in substitution of arginine by glutamine at residue 47, was revealed only in these three patients. The arginine at 47 is located between two zinc fingers and is conserved within all steroid hormone receptors. Therefore, it is highly conceivable that this amino acid substitution is responsible for the defect of the vitamin D receptor in the patients. Single-strand conformation polymorphism analysis of amplified DNA confirmed that all patients were homozygous and that parents from one family were heterozygous carriers for this mutation.     

A protein in rat prostatic interacting with androgen regulated gene

2M NaCl-insoluble fraction of rat ventral prostatechromatin(residual proteins)contain proteins able tointeract specifically with androgen-receptor complex andis,therefore,a part of the acceptor complex.Amongresidual proteins,a 97 KDa protein has been found whichbinds signifieantly to a genomic fragment containingan androgen-regulated gene coding for a 22 KDa protein.The biological significance of this binding in androgenaction need to be further studied. A mini-plasmid clone containing 22 KDa proteincoding sequence was cloned into Charon 4A genomiclibrary from which a 5.7 Kb genomic fragment wasisolated,identified by hybridization with a 5' and a 3'cDNA probes,and shown to contain the 5' flankingsequence.Restriction enzyme treatment of this fragmentyielded a 4.7 Kb restriction fragment representingthe 5' upstream region and a 1.0 Kb containing part ofthe coding sequence.Deletion studies indicated that the97 KDa protein bound only to a subclone of about 300 bpsegment.Furthermore,gel shifting experiment supportedits DNA-prptein binding.