Androgen insensitivity syndrome
The AR (MIM# 313700) is a member of the superfamily of nuclear receptors that function as ligand-dependent transcription factors. Intracellular AR is essential for androgen action, whether of testosterone or of its 5α-reduced derivative (5α-dihydrotestosterone). Hence, the AR is essential for normal primary male sexual development before birth (masculinization), and for normal secondary male sexual development around puberty (virilization). AR dysfunctions in XY individuals result in AIS (MIM# 300068)
The present version of the ARDB (available at http://androgendb.mcgill.ca) is now based on the NCBI reference sequence NM_000044.2. This is different from the original numbering scheme used over the past 20 years that was based on GenBank mRNA sequence M20132.1 [Lubahn et al., 1988]. However, a note has been added to the ARDB homepage that clearly explains the differences in the nucleotide and amino acid numbering (http://androgendb.mcgill.ca). This is so researchers can correctly identify mutations when looking up references that report AR mutations, as almost all have used the previous numbering scheme. These differences are: (1) the open reading frame starts at nucleotide 1,116 instead of 363 and (2) the variable polyglutamine tract length is two longer (23 instead of 21), whereas the variable polyglycine tract length is one shorter (23 instead of 24) for NM_000044.2 versus M20132.1, respectively. This has resulted in the AR of the new reference sequence being one amino acid longer, that is, 920aa, leading to a +1 shift in mutation numbering compared with most previously published mutations in the DNA-binding domain (DBD) and ligand-binding domain (LBD). The authors have also completed an update of the ARDB to ensure it conforms to the present HGVS standard reporting nomenclature. In order to further increase the usefulness of the ARDB, in addition to fully and partially searchable versions of the ARDB in FilemakerPro and pdf formats, we now also have an available version in Excel, and have now deposited the data into the Leiden Open Variation Database (http://www.lovd.nl/AR) [Fokkema et al., 2011].
The ARDB now contains over 800 entries of mutations causing AIS, representing over 500 different AR mutations from more than 850 patients with AIS. There has been a large increase in the number of reported AR mutations since the last published report on the database [Gottlieb et al., 2004], the number of entries rising by more than 60% from 605 to 1,029 (as on September 1, 2011). This has been partly attributed to the ease of sequencing the AR, but might easily have been even larger due to the fact that many mutations are not reported, in the literature unless they are unique. Furthermore, even unique mutations may not be reported, as they may have been found strictly in a clinical setting, as a result of sequencing of blood solely to establish a diagnosis of AIS. Until now, it has been the policy of the curator not to accept submissions to the ARDB unless they have been accepted for publication to ensure adequate quality control of the data. However, because it has become increasingly less likely that new AR mutations will be published, the home page (http://androgendb.mcgill.ca) explains that this policy has been changed. This will allow unpublished AR mutations to be included in the ARDB, provided that the curator is satisfied that the sequencing has come from qualified research or clinical laboratories. To ensure quality control, the clinical laboratories will be required to be accredited by a recognized body such as the College of Pathologists, or AABB (formerly the American Association of Blood Banks), or in Europe to be certified by EuroGeneTest as meeting the ISO 15189 standards.
While there is still an unequal distribution of the mutations along the length of the exonic regions of the AR, increasingly, new mutations are being reported that fill in areas where mutations have not been previously reported. It is also apparent that the types of mutations differ along the length of the AR. While it is still true that nearly all AIS mutations in exon 1 appear to cause complete AIS (CAIS; 89 out of 124; Table 1), since 2004 there has been an increase in mild AIS (MAIS) mutants reported (from seven to 22), which are solely due to substitution mutations. Why a significant number of missense mutations between aa 214 and 511 should cause MAIS remains a mystery, but does suggest that missense mutations in this part of exon 1 have a mild affect on AR function. However, while the total of number of exon 1 mutations (excluding those associated with CaP) has more than doubled from 54 to 124, this still represents only about 25% of the total loss-of-function mutations, despite the fact that exon 1 encodes more than half of the AR protein [Gottlieb et al., 1999]. It should be noted that this increase might partially be a reflection of the increasing ease of sequencing exon 1. What has not changed, however, is the very few AR mutants (24) that have been reported in splicing and untranslated regions of the AR (Table 1). In the C-terminal LBD, there is a striking preponderance of single-base substitution mutations, although since 2004 there has been a slight narrowing of the ratio of CAIS (144) to partial AIS (PAIS) and MAIS (110) substitution mutations (1.3/1) compared with 1.4/1 in 2004 [Gottlieb et al., 2004].
Table 1. Nature and Distribution of Unique AR Mutations that Cause Disease
|CAIS||Single-base substitution||8||28|| ||121||15||1|
| ||Premature termination||35||4||1||18|| || |
| ||Complete gene deletion||4e|| || || || || |
| ||Partial gene deletion||9||8|| ||4*|| || |
| ||Deletion (1–4 bases)||20||4|| ||10*|| || |
| ||Insertion||11||2*|| ||3*||1|| |
| ||Duplication||2||3*|| ||1*|| || |
| ||Indel|| || || ||1*|| || |
| ||Multiple-base substitution|| ||1|| ||1|| || |
| ||Premature termination||2|| || || || || |
| ||Deletion (1–4 bases)||2|| || ||2|| ||3|
|MAIS||Single-base substitution||22||4*|| ||15g|| || |
| ||Partial gene deletion|| || || ||1|| || |
| ||Deletion (1–4 bases)|| || || ||1|| || |
|Premature ovarian failure||Single-base substitution||1|| || ||2||1|| |
|Gain-of-function disease|| || || || || || ||UTR|
|Prostate cancerf||Single-base substitution||42||7||3||52|| ||2|
| ||Premature termination mutations||1|| || ||4|| || |
| ||Deletion (1–4 bases)||2||2*||1*||1||1*|| |
| ||Insertion||1*|| || || ||1||1|
|Breast cancer||Single-base substitution|| ||2|| || ||1|| |
|Larynx cancerf||Deletion (30 bases)||1|| || || || || |
|Liver cancerf||Single-base substitution||4*|| || ||1*|| || |
|Testiclular cancerf||Single-base substitution||3*|| || || || || |
The identification of specific mutations in the AR, starting in the early 1990s, as the cause of AIS has quite naturally resulted in the diagnosis of the disorder being increasingly dependent on finding AR mutations. Over the years, our laboratory has occasionally been unable to identify AR mutations in putative AIS patients that exhibit the classical AIS phenotype. Initially, the possible significance of such cases was discounted with the assumption that they were outliers, possibly because of some posttranslational event. As the number of such cases has grown, we decided to examine our own Lady Davis Institute (LDI) AIS database of AIS patients. The results were quite surprising. Out of the 75 listed patients of CAIS, no AR mutation has been identified in 25 patients, and of the 63 reported patients of PAIS, no AR mutation has been found in 37 patients (Table 2). It should be noted that all eight AR exons were sequenced from patients' genital skin and we also screened for mutations in another gene associated with an AIS-like phenotype, namely 5α-reductase 2 (SRD5A2). It is important to note that in many of these cases the diagnosis of AIS was determined before AR sequencing was available, by a detailed examination of traditional AIS-defining phenotype characteristics that included classical physical features (i.e., ambiguous external genitalia), as well as measuring androgen levels and conducting AR biochemical studies on patient-derived genital skin fibroblasts when these were available. Further, our findings are not unique, as the AIS Patient Database at the University of Cambridge, England, reports that while mutations are present in 95% of their CAIS patients, they have only been found in 25% of their PAIS patients (Dr. John Davies, personal communication). What is particularly striking about the LDI patients in whom no mutation has been identified is that a significant number have normal AR binding properties, 11 out of 25 for CAIS and 17 out 37 for PAIS patients (Table 2). This is perhaps particularly surprising in the CAIS patients, although it should be noted that in the ARDB, 30 CAIS patients are reported as having normal binding with 11 having mutations in the DBD. In summary, this suggests that at least in some of these cases, the involvement of aberrant AR protein in the AIS phenotype may not be so clear cut.
Table 2. AIS Patients from the Lady Davis Institute Database
Of particular interest is the doubling in the number of MAIS mutations (from 17 to 44), most of which are almost exclusively associated with some form of male infertility. Further, as the majority of these mutations have been found in exon 1, we have suggested that AR exon 1 mutations might be a cause of some cases of idiopathic male infertility [Gottlieb et al., 2005]. Finally, a decreasing number of mutation entries, 15% (151/1,029) as opposed to 21% (128/605) in 2004, contain data indicating the pathogenic effect of the putative mutation as the result of reconstituting the mutation and seeing the effect on AR protein function. This most probably reflects the scientific certitude that any AR mutation identified must be responsible for the AIS phenotype. However, in light of the number of patients with apparent classical AIS phenotypes that are not directly associated with an AR mutation (Table 2), perhaps it is time to at least reconsider relying largely on the presence of AR mutations to diagnose AIS.
Premature ovarian failure
Recently, a study of the AR in Indian women revealed a number of cases of onset of very early menopause, known as premature ovarian failure (POF), being associated with AR mutations [Panda et al., 2011]. Although this is just a single study, as previous attempts to identify a putative cause for POF have been unsuccessful, looking to identify AR mutations could become a promising line of investigation.
Somatic and multiple mutations
The increasing ability to sequence DNA, not just from blood cells, but also diseased tissues has led to the ability to identify mutations that are somatic, rather than germline in origin. As might be expected, the vast majority of such mutations have been found in prostate tumors and are presumed to be gain-of-function mutations (see below). The number of AIS somatic mutations identified is still relatively small, that is, 25 (Supp. Table S1), nine of which are the result of somatic mosaicism. This is perhaps due to the fact that traditionally most AR sequencing has been done using either genital skin tissue or blood, but not both. It is perhaps interesting to speculate that because almost all sequencing is now done using only blood, in some cases wherein no AR mutations have been identified, somatic mosaicism may exist, with the mutation being present solely in genital tissues or vice versa.
One of the most surprising recent developments has been the identification of individuals that have multiple AR mutations (Supp. Table S1), ranging from two to five mutations in each case. Not surprisingly, most (27/36) have been found in prostate tumors, as they are more than likely to be the result of somatic mutations. The nine cases of multiple AR mutations in AIS individuals are more difficult to explain and will be discussed later.