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Immunoreactive AR and Genetic Alterations in Subjects with Androgen Resistance and Undetectable AR Levels in Genital Skin Fibroblast Ligand-Binding Assays

Departments of Internal Medicine (D.M.A., C.M.W., N.N., J.E.G., M.J.M.) and Pharmacology (C.M.W.), University of Texas Southwestern Medical Center, Dallas, Texas 75390-8857

Address all correspondence and requests for reprints to: M. J. McPhaul, M.D., Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-8857. E-mail: michael.mcphaul{at}utsouthwestern.edu

	Abstract

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Defects of the AR cause a wide range of abnormalities of 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. This report aims to categorize the alterations of immunoreactive AR (IRAR) expression and the underlying genetic changes in a single category of patient: those in whom ligand binding is undetectable in genital skin fibroblasts. Our study found a wide range in the levels of IRAR that are detectable in fibroblast strains established from 27 such individuals. A large proportion (19 of 27) express significant amounts of AR protein, as detected using a sensitive Western blot technique. In a smaller number (8/27), AR expression was undetectable. Intact IRAR was identified in16 of the 19 fibroblast strains in which AR expression could be detected. The AR gene was analyzed in 14 strains from this group. In 13 instances, single amino acid substitutions were identified within the ligand-binding domain of the receptor protein. In three of the remaining patients (3 of 19), truncation of the receptor protein was suggested by the rapid migration of the IRAR in SDS-polyacrylamide gels. In those three patients, production of the shortened immunoreactive receptor was traced to mutations that interrupted the AR open reading frame. By contrast, only one of the eight patient samples with no detectable IRAR carried a mutation that resulted in a single amino acid substitution. An interruption of the AR open reading frame was identified in six of the eight strains in which immunoreactive receptor was absent. In the remaining strain, no mutation was present within or surrounding the eight coding exons. This study serves to define the effects that mutations of the AR have on the levels of expressed immunoreactive receptor protein. In addition, it demonstrates the type of information that can be obtained if an immunoblot assay were to be used as a component of a screening method to analyze samples from patients with defects of the AR. Finally, the study suggests that in some androgen-resistant patients, defects outside the AR open reading frame may result in major alterations of AR expression.

	Introduction

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NORMAL MALE SEXUAL development requires the expression of a functional AR protein and the synthesis by the testes of the major circulating androgens, T, and 5-dihydrotestosterone. The ability to form an active complex between AR and androgen is critical to the modulation of genes regulated by androgen. Thus, the regulation of genes by androgen may be disturbed by a variety of genetic alterations that influence the structure or stability of the AR as well as alterations that interfere with the normal synthesis of the hormones themselves (1, 2, 3).

In common with other members of the nuclear receptor family, the AR protein molecule contains four main functional segments: an amino (N-) terminal region that is involved in transcriptional regulation, a DNA-binding domain, a hinge region containing the nuclear-targeting sequence, and a carboxy (C-) terminal ligand-binding domain (LBD) (4, 5). Two distinct AR isoforms have been detected in human cells and tissues. The full-length AR-B isoform appears in immunoblots as a 110- to 112-kDa doublet recognized by antibodies to the N terminus (amino acids 1–20) and antibodies to an internal peptide (internal A: amino acids 200–220) of the human AR. A shorter isoform (AR-A) lacking the N-terminal epitope appears as a single 87-kDa band and is detected by antibodies that recognize the internal A sequence. AR-B is the predominant form of AR protein found in fibroblast strains derived from normal individuals and in all human tissues examined to date (6, 7).

Owing to the location of the AR gene on the X chromosome, 46,XY individuals possess only a single AR gene. Therefore, any mutation in the AR gene that alters the activity of the AR protein would effect the development and function of all androgen-sensitive tissues of the individual. A variety of molecular defects in the AR gene have been reported including complete or partial gene deletions, base pair deletions or insertions, and trinucleotide repeat expansions. Some of these mutations may then encode premature termination codons, result in missense mutations, or alter mRNA splicing. Failure to form an active AR-ligand complex or failure of the complex to interact with the regulatory regions of androgen-responsive genes would be expected to render androgen target organs resistant to hormone action. Not surprisingly, many of the mutations associated with human androgen insensitivity syndromes identified to date produce changes in either the ligand-binding or DNA-binding domains of the AR molecule (8, 9).

A wide spectrum of phenotypic abnormalities in sexual development and virilization is seen in AIS patients with androgen insensitivity syndromes (1, 8). Studies of ligand binding in cultured genital skin fibroblasts derived from many androgen-resistant individuals have revealed that reduced binding levels and/or qualitatively different binding characteristics are frequently associated with amino acid substitutions in the ligand-binding domain. Mutations in the AR DNA-binding domain have been reported for some androgen-resistant individuals whose fibroblasts display normal levels of ligand binding. However, in other cases, no changes in the AR gene coding sequence have been identified by nucleotide sequence analysis, raising the possibility that androgen insensitivity in these individuals may involve postreceptor defects or factors that regulate transcription of the AR gene (10, 11).

To date, the complete absence of ligand binding in genital skin fibroblasts invariably has been associated with the testicular feminization phenotype characteristic of X-linked complete androgen insensitivity syndrome (CAIS). However, the ligand-binding assay does not indicate whether AR protein is absent in such patients, originally termed receptor negative, or whether they produce an abnormal form of AR that is unable to bind ligand. To investigate the frequency and mechanism of this defect, we have used an immunoblot assay to determine the quantity and apparent molecular size of AR-B proteins present in fibroblasts derived from 27 CAIS patients with absent ligand binding. In parallel, we have correlated the results of these immunoblot analyses with the genetic studies of 25 of these patients.

	Materials and Methods

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Clinical history and phenotype

Various physicians referred the patients described in this study. Phenotype was established by descriptions provided by the referring physicians using established criteria (1). Previous studies have identified AR mutations in seven of these subjects, as indicated in the table and discussed in the text (12, 13, 14, 15, 16).

Cell culture and sample preparation

Primary cultures of genital skin from androgen-resistant patients and from a control subject (strain 704) were established as previously described (17) and maintained in DMEM with 20% FCS and 1% penicillin and streptomycin. Samples for immunoblot analysis were obtained by growing cells to confluence, harvesting and extracting with SDS-PAGE loading buffer as reported elsewhere (14). All protocols and consent forms were reviewed and approved by the Institutional Review Board at University of Texas Southwestern Medical Center.

Monolayer binding assays

Specific androgen-binding capacity was determined as reported previously (17). In brief, fibroblast monolayers were incubated with 0.25–3.0 nM [³H]-dihydrotestosterone in the presence or absence of a 500-fold excess of unlabeled ligand, harvested with trypsin, and disrupted by sonication. Aliquots were assayed for radioactivity and protein. Samples are defined as absent ligand binding when less than 4 fmoles of specific binding of [³H]-dihydrotestosterone is detected (1, 17).

Immunoblot assays

Immunoblot assay samples were subjected to SDS-PAGE fractionation in standard 7.5% acrylamide gels, transferred to nitrocellulose membranes, and incubated sequentially with polyclonal antipeptide antibodies that recognize the N-terminal 20 amino acid sequence of AR and with ¹²⁵I labeled antirabbit IgG. Immunoreactive AR (IRAR) bands were visualized by autoradiography and quantified by densitometry as described previously (14). The relative level of immunoreactivity per fibroblast cell was determined by comparing the density of the 110- to 112-kDa doublet with a standard curve obtained by including samples of a standard preparation of the normal control strain 704 in each assay. The IRAR level in the control standard, set at 100%, was equal to the average value obtained from 12 different preparations of 704 fibroblasts. Aliquots of the control standard were included in each of the immunoblots, permitting the levels of IRAR present in individual samples to be estimated relative to the amount present in 704 fibroblasts. This method has been standardized and applied to a set of normal subjects and a group of patients with qualitative abnormalities of the AR (14). Samples that did not react with anti-N-terminus antibodies were also tested with antibodies that recognize the 200–220 amino acid (internal A) sequence of human AR to determine whether the lack of immunoreactivity was because of deletion of the N terminus.

PCR analysis of CAG/CAA repeat length

The AR gene segment in exon 1 encoding a glutamine (Gln) homopolymeric tract was amplified using PCR primers spanning the CAG/CAA repeat region (18). The resulting PCR products were separated on 5% NuSieve gels (BMA, Rockland, ME). The number of CAG/CAA repeats was estimated by comparison with a sequencing ladder and with PCR products obtained with DNA from fibroblast strains with known nucleotide sequences or from cells transfected with expression plasmids encoding androgen receptors with 12, 20, or 46 residues in the Gln homopolymeric segment. Construction of these expression plasmids has been described (19).

AR gene structure

Genomic DNA was prepared and the AR gene structure was analyzed by amplifying individual exon segments, followed by nucleotide sequence analysis, as described previously (16). For those samples in which immunoreactive AR was measurable, exons 3–8 were amplified. In those instances in which no IRAR was detected, exons 1–8 were amplified and sequenced.

	Results

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Detection of immunoreactive AR protein in fibroblasts with absent ligand-binding capacity

The representative immunoblots presented in Fig. 1 demonstrate that as little as 2% of the immunoreactive AR-B protein present in the standard control strain (strain 704) can be detected using this assay. The immunoblots presented in Fig. 1 include genital skin fibroblast samples derived from 12 individuals with androgen resistance. In each instance, androgen binding was undetectable in ligand-binding assays. Antibodies to the N-terminal 20 amino acid sequence (Fig. 1A) reveal that three of these patients (902, 827, and 961) produce the characteristic 110- to 112-kDa AR-B doublet found in normal control fibroblasts (704). Antibodies to the internal A 200–220 amino acid sequence (Fig. 1B) recognize both AR-B and the 87-kDa AR-A band in the control and samples from three other patients (895, 909, and 105). However, neither antibody preparation detected any IRAR in samples from the six patients included in both assays.

View larger version (60K): [in this window] [in a new window]   Figure 1. Immunoblot analysis of immunoreactive AR protein levels expressed in genital skin fibroblasts derived from 12 patients who lack ligand-binding capacity and from a normal subject (704). Fibroblast strains are identified by numbers at the top of each lane. Numbers in parentheses indicate the number of fibroblast cells (x 104) represented by each sample. Positions and sizes of 69-kDa and 97-kDa molecular mass (MW) markers are indicated by bars on the left side of the blot. Positions of the 110- to 112-kDa AR-B doublet and 87-kDa AR-A band are indicated on the right. A, Antibodies raised to the amino terminal region of the human AR protein recognize the AR-B isoform in 827, 902, and 961 fibroblasts and detect AR-B in samples of the standard 704 preparation that represent as few as 2 x 104 fibroblast cells. B, Antibodies raised to the 200–220 amino acid sequence (internal A) of the human AR protein recognize both AR-A and AR-B isoforms in 105, 895, and 909 fibroblasts and in the standard 704 preparation. Neither antibody preparation detects immunoreactive AR bands in fibroblasts from the other six fibroblast strains included in these experiments.

View larger version (66K): [in this window] [in a new window]   Figure 2. Immunoblot analysis detects a truncated AR in the 997 fibroblast strain. The IRAR protein expressed in the 997 fibroblast strain is compared with the normal 110- to 112-kDa AR-B doublet that is detected in 704 normal control fibroblasts and the smaller AR doublet that is present in 717 fibroblasts. Strain 717 carries a mutation that results in premature termination of the AR protein (14 ). Differences in mobility on SDS-PAGE were examined by running fibroblast extract samples side by side in the same gel, alone or in combination. Numbers in parentheses indicate the amount of protein (micrograms) applied to each lane. To compensate for differences in immunoreactive AR levels between strains while applying similar amounts of total protein per lane, the 704 control preparation was diluted 1:4 with extracts from a strain that does not produce the AR-B isoform (strain 776, Ref.6 ) or from 717 or 997, both strains that produce relatively low levels of IRAR protein. The AR species found in 997 fibroblasts is similar in mobility to the truncated form of AR produced by strain 717.

As indicated in Table 1, however, not all of the samples from this group of individuals contained detectable AR protein. The 110- to 112-kDa AR protein doublet was not detected in fibroblasts derived from eight other CAIS patients, including one pair of siblings (subjects 694 and 867). A very faint single approximately 108-kDa band (too weak to quantify) appeared occasionally in immunoblots of sample 1017. The significance of this observation is uncertain.

Size of IRAR proteins present in negative-binding fibroblasts that express the AR gene

The immunoreactive bands detected in most fibroblast strains in this study appeared at the same position as the AR-B doublet from the 704 standard (control strain) as demonstrated in Fig. 1, indicating little or no difference in molecular mass. However, three patients (717, 750, and 997) produce AR that migrates faster in standard gels than the normal full-length AR-B protein (14, 15 and Fig. 2). The immunoblot presented in Fig. 2 demonstrates the difference in apparent molecular mass of AR proteins produced by 717 and 997 fibroblasts, compared with the standard 110- to 112-kDa AR expressed in the control strain 704. This comparison indicates that the AR species produced by patient 997 is similar in size to the truncated AR protein produced by patient 717. Although this blot was probed with antibodies to the internal A region of the protein, similar results were obtained using anti-N-terminal antibodies, showing that the N terminus of the AR is intact in the AR expressed in the 997 fibroblasts (data not shown).

Structures of the AR genes of patients with absent ligand binding in monolayer binding assays

Normal-size IRAR. Immunoblot analysis of fibroblast extracts prepared from the majority (16 of 19) of patients in this group detected variable amounts of AR that migrated with an apparent molecular mass of approximately 110–112 kDa. Although the level of specific androgen binding was undetectable in all samples, the quantity of immunoreactive receptor that was detected varied considerably. PCR amplification of exons 3–8 revealed bands of the expected size in all instances. Sequence analysis of the cloned fragments revealed a variety of different nucleotide substitutions within the LBD of receptor protein. In all but one instance (strain 836), a single amino acid replacement was localized to the LBD of receptor protein. These results are summarized in Table 1.

Altered-size IRAR. Fibroblast strains derived from three CAIS patients (717, 750, and 997) produced IRAR proteins with abnormally rapid electrophoretic mobility that could not be accounted for on the basis of changes in the size of the Gln repeat sequence (Fig. 3). In addition, bands of the same size were detected when antibodies directed at distinct epitopes within the amino terminus (anti-internal A) were employed. These findings suggested truncation at or near the carboxyl terminus of the receptor open reading frame. This prediction has been confirmed by the identification of mutations that interrupted the receptor open reading frame. In two instances (717 and 750; Refs.14 and15 , respectively), single nucleotide substitutions introduced a premature termination codon. In the third instance (997), a single nucleotide deletion leads to a frame shift of the open reading frame with subsequent premature termination (Table 1).

View larger version (66K): [in this window] [in a new window]   Figure 3. Agarose gel electrophoresis of PCR products obtained by amplification of the CAG/CAA repeat segment in exon 1 of the AR gene. The size of the repeat sequence in eight patients that lack androgen-binding capacity was estimated by comparing the position of PCR products obtained with DNA extracted from fibroblast strains derived from these individuals; strains (704, 787, 814, and 980) with known nucleotide repeat sequences (6 21 ); or transfected cells carrying AR genes encoding 12, 20, or 46 residues (identified as Gln12, Gln20, and Gln46). All of the strains tested in this experiment carried repeat sequences that are within the range (11–31) found in an unaffected control group (20).

In four instances, somewhat unexpected results were obtained. In two strains established from related affected subjects (694 and 867), exon 2 could not be amplified from genomic DNA, suggesting an alteration of the structure of this region of the AR gene. Equally unexpected was the identification of a single amino acid substitution in strain 958 (serine⁸⁸⁸ lysine). Finally, in one case (strain 183) no sequence abnormality could be identified within the AR open reading frame (Table 1).

A flow diagram of the different sample groups and analyses is shown in Fig. 4. A summary of the genetic alterations identified is presented in Fig. 5.

View larger version (17K): [in this window] [in a new window]   Figure 4. Summary of the immunoblot and genetic analyses. The results of the immunoblot and AR gene sequence analyses are presented as a flow diagram. Twenty-seven fibroblast strains established from CAIS patients were analyzed using a sensitive immunoblot assay. In subsets of this group of samples, normal-size or truncated IRAR was detected. In a smaller proportion, no IRAR was detected. The results of the genetic analyses are summarized for the individual categories. NAI, No abnormality identified.

View larger version (19K): [in this window] [in a new window]   Figure 5. Schematic representation of the genetic abnormalities identified. Upper panel, A summary of the mutations identified in fibroblasts in which IRAR was measurable. In most instances, single amino acid substitutions were identified in those strains in which normal-size AR was detected in immunoblots. In a small proportion of samples, a truncated receptor protein was identified. In this latter group of samples, point mutations leading to an interruption of the AR open reading frame were identified. Lower panel, A summary of the mutations identified in fibroblasts in which AR could not be detected using sensitive immunoblot assays. In four instances, mutations were identified that predict an interruption of the AR open reading frame. It is presumed that the AR proteins synthesized in these strains are unstable and do not accumulate to levels that could be detected in the immunoblot assays. In two related strains, exon 2 could not be amplified and these patients are presumed to harbor a deletion of all or part of exon 2 (indicated by the bold bar).

	Discussion

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The majority of patients included in this report (19 of 27) expressed detectable levels of AR as determined using a sensitive immunoblot assay. In 17 of these 19 patients, genetic analyses were performed to identify the causative lesion.

In 16 of these 19 patients, normal-sized AR was detected. Single amino acid substitutions within the ligand binding were identified in 13 of the 14 samples in this category that were analyzed at the genetic level. In one case (patient 836), a single nucleotide deletion was identified, which resulted in frame shift alteration of the receptor. It is presumed that in this instance, the amount of normal-sized receptor that accumulates reflects the small proportion of the unmutated mRNA found in this strain (see Table 1).

In 3 of the 19 samples analyzed in this category, immunoreactive receptor was identified that migrated with a mobility faster than that of the normal full-length AR. In each of these instances, termination codons were identified within the AR open reading frame caused either by the insertion of single point mutations (strains 717 and 750) or as the result of a frame shift mutation (strain 997).

In a smaller proportion of patients within this group (8 of 27), no IRAR was detected using our sensitive immunoblot method. Analysis of the structure of the AR genes in these patients revealed a variety of genetic alterations. In one case (strain 429), a premature termination codon is introduced as a result of a single nucleotide substitution. In two other cases (strains 942 and 1111), premature termination occurs as a result of a frame shift mutation in the open reading frame. In like fashion, the open reading frame is disrupted in strain 1017 as a result of a mutation at the splice acceptor site at the 5' boundary of exon 4. In each instance, it is presumed that the truncated proteins resulting from these mutations are unstable or are inefficiently synthesized, leading to undetectable levels of the truncated receptor protein. In four strains within this category, unexpected results were encountered. In two affected siblings (patients 694 and 867), it proved impossible to amplify exon 2 using a variety of different oligonucleotide primers. These results suggest the possibility that exon 2 has undergone a major structural alteration in this particular pedigree. In strain 958, no mutations were identified that interrupted the integrity of the open reading frame. Instead, only a single amino acid substitution (serine⁸⁸⁸ lysine) was identified. At present, it is unclear whether this substitution is adequate to explain the undetectable levels of AR in this strain or whether this is simply a polymorphism associated with another alteration of the AR gene that leads to the absence of AR expression. Finally, no mutations were identified in strain 183 within the entire open reading frame of the AR gene. This suggests that alterations outside the AR open reading frame may have contributed to the absence of AR in this strain.

In summary, we have examined the levels of IRAR expression in 27 fibroblast strains in which ligand binding is undetectable in fibroblast monolayer-binding assays. This Western blot analysis provided information regarding both the level and apparent molecular size of the AR expressed. Correlation of this information with results from an analysis of the AR gene was possible in 25 subjects. The identification of normal-sized AR was most frequently associated with the identification of the genetic alteration resulting in a single amino acid substitution within the LBD (13 of the 14 analyzed at the genetic level). The visualization of a truncated receptor protein was traced to mutations causing premature termination of receptor protein (3/3). Finally, the absence of IRAR was associated with a variety of mutations that interrupt the AR open reading frame (termination codons [1]), frame shift mutations [2], partial gene deletions [2], or alterations of mRNA processing [1]). Only one of the eight patients with undetectable IRAR carries a mutation that results in a single amino acid substitution. In one strain, no alterations were identified with or surrounding the AR reading frame. These results suggest that in most instances a combination of an immunoblot analysis and an assay to measure AR function would permit the type of AR mutation to be deduced correctly.

	Acknowledgments

 
We thank Diane Allman for culturing the cells used in this study. Kailas Patel, Nancy Stallings, and Nicole Tarrant provided excellent technical support.

	Footnotes

 
This work was supported by Grant DK-03892 from the NIH and Grant I-1090 from The Robert A. Welch Foundation. Portions of this work were presented at the 75th Annual Meeting of The Endocrine Society, Las Vegas, Nevada, June 9–12, 1993.

Abbreviations: CAIS, Complete androgen insensitivity syndrome; Gln, glutamine; IRAR, immunoreactive AR; LBD, ligand-binding domain.

Received June 29, 2001.

Accepted October 4, 2001.

	References

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