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.     

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.     

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.     

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.     

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.     

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.     

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.     

The androgen receptor gene mutations database.

The current version of the androgen receptor (AR) gene mutations database is described. We have added (if available) data on the androgen binding phenotype of the mutant AR, the clinical phenotype of the affected persons, the family history and whether the pathogenicity of a mutation has been proven. Exonic mutations are now listed in 5'-->3' sequence regardless of type and single base pair changes are presented in codon context. Splice site and intronic mutations are listed separately. The database has allowed us to substantiate and amplify the observation of mutational hot spots within exons encoding the AR androgen binding domain. The database is available from EML (ftp://www.ebi.ac.uk/pub/databases/androgen) or as a Macintosh Filemaker file (MC33@musica.mcgill.ca).     

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.     

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.     

A novel mutation in the D-box of the androgen receptor gene (S597R) in two unrelated individuals Is associated with both normal phenotype and severe PAIS

BACKGROUND: An absent or dysfunctional androgen receptor (AR) in 46,XY individuals is the most common cause of various degrees of undermasculinization. Therefore, we routinely perform sequencing of the AR gene in all cases with suspected androgen insensitivity. METHODS: In a newborn 46,XY male diagnosed with partial androgen insensitivity syndrome and a phenotypically normal man, who in childhood had bilateral cryptorchidism, the AR was directly sequenced. Seven additional men with cryptorchidism in infancy were chosen as controls. RESULTS: An AR variant (S597R) was identified in both males. Treatment of the newborn with 1% dihydrotestosterone ointment locally, resulted in normal penile size for age. Sequencing of the region in 7 other men with cryptorchidism in infancy did not reveal any additional deviation from the normal reference sequence. CONCLUSION: The same mutation at this codon can cause significantly different phenotypes as shown by the variation in masculinization of these individuals, with 1 severely affected child and 1 normally developed man. However, the S597R mutation does not seem to be a common cause of undescended testes in boys. Despite the S597R mutation and severe undermasculinization, as seen in the baby, normal male phenotype for age could be achieved with treatment.     

A mutation in the ligand binding domain of the androgen receptor of human LNCaP cells affects steroid binding characteristics and response to anti-androgens

LNCaP prostate tumor cells contain an abnormal androgen receptor system. Progestagens, estradiol and anti-androgens can compete with androgens for binding to the androgen receptor and can stimulate both cell growth and excretion of prostate specific acid phosphatase. We have discovered in the LNCaP androgen receptor a single point mutation changing the sense of codon 868 (Thr to Ala) in the ligand binding domain. Expression vectors containing the normal or mutated androgen receptor sequence were transfected into COS or Hela cells. Androgens, progestagens, estrogens and anti-androgens bind the mutated androgen receptor protein and activate the expression of an androgen-regulated reporter gene construct (GRE-tk-CAT). The mutation therefore influences both binding and the induction of gene expression by different steroids and antisteroids.