It has been known for quite some time that defects in male sexual differentiation in 46, XY individuals have an X-linked pattern of inheritance. It was Reifenstein who reported in 1947 on families with severe hypospadias, infertility and gynecomastia (83 ). The end-organ resistance to androgens has been designated as androgen insensitivity syndrome (AIS) and is distinct from other forms of male pseudohermaphroditism like 17β-hydroxy-steroiddehydrogenase type 3 deficiency or 5α-reductase type 2 deficiency (2, 84, 85 ). It is generally accepted that defects in the androgen receptor gene can prevent the normal development of both internal and external male structures in 46, XY individuals and information on the molecular structure of the human androgen receptor gene has facilitated the study of molecular defects associated with androgen insensitivity. Due to the X-linked character of the syndrome, only 46, XY individuals are affected, while in female carriers only sporadic reports are available on delayed menarche (86 ). Naturally occurring mutations in the androgen receptor gene are an interesting source for the investigation of receptor structure-function relationships. In addition, the variation in clinical phenotypes provides the opportunity to correlate a mutation in the androgen receptor structure with the impairment of a specific physiological function.

The main phenotypic characteristics of individuals with the complete androgen insensitivity syndrome (CAIS) are: female external genitalia, a short, blind ending vagina, absence of wolffian duct derived structures like epididymides, vasa deferentia and seminal vesicles, the absence of a prostate, the absence of pubic and axillary hair and the development of gynecomastia (87, 88). Müllerian duct derived structures are usually absent because anti-müllerian hormone action is normal due to the presence of both testes in the abdomen or in the inguinal canals. Usually, testosterone levels are within the normal range ( 10 - 40 nmol/L) or elevated, while elevated luteinizing hormone (LH) levels (> 10 IU/L) are also found indicating androgen resistance at the hypothalamic-pituitary level. The high testosterone levels are also substrate for aromatase activity, resulting in substantial amounts of estrogens, which are responsible for further feminisation in CAIS individuals.

In the partial androgen insensitivity syndrome (PAIS) several phenotypes ranging from individuals with predominantly a female appearance (e.g. external female genitalia and pubic hair at puberty, or with mild cliteromegaly, and some fusion of the labia) to persons with ambiguous genitalia or individuals with a predominantly male phenotype (also called Reifenstein syndrome) (87, 88 ). Patients from this latter group can present with a micropenis, perineal hypospadias, and cryptorchidism. In the group of PAIS individuals, wolffian duct derived structures can be partially to fully developed, depending on the biochemical phenotype of the androgen receptor mutation. At puberty, elevated luteinizing hormone, testosterone, and estradiol levels are observed, but in general, the degree of feminization is less as compared to individuals with CAIS. Individuals with mild symptoms of undervirilization (mild androgen insensitivity syndrome) and infertility have been described as well.Phenotypic variation between individuals in different families has been described for several mutations (88, 89, 90, 91). However, in cases of CAIS no phenotypic variation has been described within one single family, in contrast to families with individuals with PAIS (92).

Since the cloning of the androgen receptor cDNA in 1988 and the subsequent elucidation of the genomic organization of the androgen receptor gene, molecular biology tools have been available for the molecular analysis of the androgen receptor gene in individuals with AIS [Figure 7, see above] (28, 29). In addition to endocrinological data such as levels of testosterone, luteinizing hormone, androstenedione, and 5α-dihydrotestosterone, which can vary widely in AIS individuals, the most reliable approach is the sequencing of each individual androgen receptor exon and the flanking intron sequences. In general, AIS can be routinely analyzed and separated from entirely different syndromes presenting with similar phenotypes including testicular enzyme deficiencies, 5α-reductase type 2 deficiency, Leydig cell hypoplasia due to inactivating luteinizing hormone receptor mutations. Furthermore, in pedigree analysis intragenic polymorphisms like the highly polymorphic (CAG)nCAA repeat encoding a poly-glutamine stretch, the polymorphic GGN repeat encoding a poly-glycine stretch, the HindIII polymorphism [Figure 8, see above] (26) and the StuI polymorphism (89), can be used as X-chromosomal markers (38, 94, 95). Extensive general information can be obtained at the internet site: www.genecards.org on the AR (NR3C4) gene and on the 233 identified single nucleotide polymorphisms (SNP’s).

In the androgen receptor gene, 4 different types of mutations have been detected in 46, XY individuals with AIS: single point mutations resulting in amino acid substitutions or premature stop codons, nucleotide insertions or deletions most often leading to a frame shift and premature termination, complete or partial gene deletions (>10 nucleotides), and intronic mutations in either splice donor or splice acceptor sites which affect the splicing of androgen receptor RNA (96 ). In general in 70% of the cases, AR gene mutations are transmitted in an X-linked recessive manner, but in 30% the mutations arise de novo. When de novo mutations occur after the zygotic stage, they result in somatic mosaicisms (97). The most recent update on androgen receptor gene mutations is available at http://www.mcgill.ca/androgendb/(96).

Mutations in the NH2-terminal domain (exon 1 of the gene) do not occur frequently and the vast majority of the mutations result directly in a stop codon or in premature termination due to frameshifts caused by nucleotide insertions or deletions ( http://www.mcgill.ca/androgendb/). Mutations in 41 different codons have been reported in the NH2-terminal domain, which is in approx. 8% of all codons in exon 1.

An interesting mutation is described in the fourth nucleotide, which results in a decreased translational efficiency of the androgen receptor mRNA in an individual with PAIS (98 ). Three other missense mutations were reported in combination with mosaicism or with a mutation in an other region of the gene.In a family with PAIS associated with severe hypospadias, the length of the androgen receptor NH2-terminal poly-glutamine repeat has been reported to be shortened to only 12 glutamine residues (99). The shortened glutamine stretch as such, is not the cause for the androgen resistance but seems to increase the thermolability of the androgen receptor in combination with a point mutation in exon 5 (Y763C) in the ligand binding domain. This point mutation causes rapid dissociation but no thermolability. These data support a functional interaction of the two separated regions in the androgen receptor and indicates further that the defect becomes critical in only part of the androgen target tissues because of the partial character of the androgen resistance found in this family (99).

In general, mutations in the DNA binding domain (e.g. single nucleotide substitutions) result in a normal hormone-binding protein, which is defective in DNA-binding/dimerization and consequently in transcription activation. In total 46 different mutations have been reported in 30 different codons in the DNA-binding domain, which is in approx. 27% of all codons in exons 2 and 3 ( http://www.mcgill.ca/androgendb/). Twenty-one mutations were observed in the first zinc cluster and twenty-one in the second zinc cluster. Since the 3D structures of the DNA-binding domain of several nuclear receptors have been published, the consequence of mutations in the androgen receptor DNA-binding domain can predicted on basis of the structure of the glucocorticoid receptor DNA-binding domain (60). This is illustrated in two studies in which 3D-modelling of the mutated DNA binding domain of the androgen receptor predicts the functional activity of mutant receptors (100, 101 ). A mutation (G577R) in the so-called P-box [Figure 10, see above], which is involved in androgen response element recognition, was found in a PAIS individual. This mutation affected differentially transactivation of several natural and synthetic promoters, suggesting that androgen target genes may be differentially affected by this mutation (102). An interesting observation was made with respect to the second zinc cluster in which either one of two adjacent arginine residues (Arg607 & Arg608) were found to be mutated in PAIS individuals who developed breast cancer [Figure 10, see above] (103, 104 ). It is speculated that a decrease in androgen action within the breast cells could account for the development of male breast cancer by the loss of a protective effect of androgens. However, the same mutations in several other PAIS individuals did not result in breast cancer development.

The mutation Ala596Thr in the second zinc cluster in the so-called D-box resulted in abolishment of dimerization in a PAIS individual [Figure 10, see above] (105 ). A similar mutation at an identical position in the second zinc cluster of the glucocorticoid receptor DNA-binding domain has been created to discriminate between dimerization/DNA binding of the glucocorticoid receptor and protein-protein interactions with other transcription factors such as the AP-1 transcription complex (106 ). It appeared that the dimerization mutant did not affect the cross-talk with other transcription factors. In this way, a tissue specific response can be influenced by a single amino acid change and if this is also true for the mutant androgen receptor then the partial phenotype can be explained. Interestingly a Ser579Arg , also located in the D-box can cause significantly different phenotypes ranging from under-virilisation to a normal male phenotype (107).

In the so-called hinge region, located between amino acid residues 622 and 670 [Figure 9, see above], only six mutations have been reported. The relatively low number of mutations in the hinge region (only in 8% of all codons) indicates that this region might be very flexible and that some variation in composition and length of this region is not detrimental for androgen receptor function http://www.mcgill.ca/androgendb/.

Two amino acid substitutions within the hinge region have been described that resulted in CAIS and three in PAIS. The I664N substitution on the border of the hinge region and ligand-binding domain, resulted in a decreased hormone binding (108 ).

It can be expected that mutations in the ligand binding domain might affect different functional aspects (eg. loss of ligand binding, changes in ligand binding affinity and specificity, changes in co-activator receptor interactions, changes in receptor stability and thermolability). A large number of mutations (222 different mutations in 139 codons, which is in 56 % of all codons of the ligand binding domain) in the ligand binding domain have been reported in all 5 exons in individuals with either CAIS or PAIS ( http://www.mcgill.ca/androgendb/). However, it appears that most mutations are located in exon 4 (28 mutations in helix 3), in exon 5 (32 mutations in helices 4 and 5) and in exon 7 (31 mutations in helices 9 and 10; and β strands). Interestingly mutations are found in 13 of the 18 amino acid residues considered to interact with the ligand directly (62). For some mutations (in total 12, distributed over the whole ligand binding domain) both a complete and a partial phenotype has been described, indicating that phenotype does not always match with genotype. In the AF-2 core region (893-EMMAEIIS-900) of the androgen receptor ligand-binding domain a relatively low number of mutations have been reported [Figure 9, see above]. Only at positions M895 and Ile898 mutations have been described in individuals with the complete syndrome (109 110 ). It can be speculated that in this part of helix 12 mutations in the androgen receptor ligand-binding domain are less deleterious for androgen receptor function as compared to those in helix 5 and in the β-turn, where almost every amino acid residue has been found to be mutated in AIS individuals. However, functional analysis of a novel AR mutation, Q902K in helix 12, in an individual with partial androgen insensitivity, indicated that this residue is important for modulation of NH2/COOH terminal interaction and TIF-2 activation (111).

Only a few cases have been reported on partial or complete androgen receptor gene deletions, indicating the relatively low frequency of this type of androgen receptor defect (112, 113, 114, 115, 116, 117, 118 ) ( http://www.mcgill.ca/androgendb/). All cases reported are found in CAIS individuals, with the exception of two cases, one in which an exon 4 deletion was found in a person with azoospermia (113 ) and another one in which a large intron 2 deletion of at least 6 kb was reported involving a branch point site, which resulted in a partial exon 3 skipping during the splicing process (117).

Deletion of either exon 3 or exon 4 occur both in-frame and result in a non-functional protein lacking either the second zinc cluster or the hinge region and the NH2-terminal part of the ligand-binding domain [Figure 7, see above]. In case of an exon 3 deletion, an intact and functional ligand-binding domain is present [Figure 7, see above]. So far, functionally significant mutations in the androgen receptor promoter region or in the 5'- and 3'- untranslated regions of the gene have not been reported.

A special group of interesting, but rare mutations are the splice donor and splice acceptor site mutations in the androgen receptor gene in AIS individuals ( http://www.mcgill.ca/androgendb/). For all splice donor sites in the gene, the consensus splice donor site sequence GTAAG/A is present. The 8 reported mutations in donor splice sites are all substitutions either at position +1 (G --> A or G --> T), position +3 (A --> T), position + 4 (A --> T) or position + 5 (G --> A) and result in defective splicing with the consequence of one or more exons spliced out, or the use of a cryptic splice donor site within the preceding exon (108, 109, 119, 120, 121, 122 ). In 7 of the reported cases, the phenotype is complete androgen insensitivity. In one case, an insertion of one nucleotide (T) at position + 4 in the splice donor site of intron 6 has been reported, resulting in a partial androgen insensitive phenotype (123 ). Only 3 mutations have been reported in splice acceptor sites, which all affect the splicing of the androgen receptor RNA. Interestingly, a substitution at position -11 (T>G) has been found in the pyrimidine-rich region of the splice acceptor site of intron 2, resulting in the activation of a cryptic splice acceptor site at position -70/-69 and consequently in the insertion of 69 nucleotides (corresponding to 23 additional amino acid residues) in the mRNA between exons 2 and 3 (73). The corresponding protein is defective in DNA-binding because the insertion has occurred between the first and second zinc cluster [Figure 7, see above].