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Complete androgen insensitivity syndrome caused by a deep intronic pseudoexon-activating mutation in the androgen receptor gene

View Article: PubMed Central - PubMed

ABSTRACT

Mutations in the X-linked androgen receptor (AR) gene underlie complete androgen insensitivity syndrome (CAIS), the most common cause of 46,XY sex reversal. Molecular genetic diagnosis of CAIS, however, remains uncertain in patients who show normal coding region of AR. Here, we describe a novel mechanism of AR disruption leading to CAIS in two 46,XY sisters. We analyzed whole-genome sequencing data of the patients for pathogenic variants outside the AR coding region. Patient fibroblasts from the genital area were used for AR cDNA analysis and protein quantification. Analysis of the cDNA revealed aberrant splicing of the mRNA caused by a deep intronic mutation (c.2450-118A>G) in the intron 6 of AR. The mutation creates a de novo 5′ splice site and a putative exonic splicing enhancer motif, which leads to the preferential formation of two aberrantly spliced mRNAs (predicted to include a premature stop codon). Patient fibroblasts contained no detectable AR protein. Our results show that patients with CAIS and normal AR coding region need to be examined for deep intronic mutations that can lead to pseudoexon activation.

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Expression of AR mRNA (a), androgen target gene FKBP5 (b), and AR protein (c) in fibroblasts and LNCaP prostatic cancer cells. The cells were split onto 6-well plates, and after 24 h, the medium was changed to steroid-depleted medium for 6 h. A half of the wells were treated with vehicle (0.1% ethanol) (−) and a half with 1 nM R1881 (+) for 18 h before immunoprecipitation or RNA extraction. Samples XX31B, XY31A, and XX54A are control fibroblasts. The patient-derived fibroblasts are samples T1 and T2. GAPDH served as the reference gene for quantification of AR and FKBP5 mRNA (panels a and b). The expression of all samples was normalized to the control sample XX31B vehicle treatment. The bars represent mean ± SD of 3–5 independent samples. In (c), AR was immunoprecipitated with rabbit polyclonal α-AR17 and detected in western blotting with mouse monoclonal α-AR 441 recognizing AR amino acids 299–315 in the AR N-terminal domain. α-GAPDH was used to control the loading of input samples. The samples in the different blots are from the same experiment. The blots have been cropped; full-length blots are presented in Supplementary Figure S1.
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f2: Expression of AR mRNA (a), androgen target gene FKBP5 (b), and AR protein (c) in fibroblasts and LNCaP prostatic cancer cells. The cells were split onto 6-well plates, and after 24 h, the medium was changed to steroid-depleted medium for 6 h. A half of the wells were treated with vehicle (0.1% ethanol) (−) and a half with 1 nM R1881 (+) for 18 h before immunoprecipitation or RNA extraction. Samples XX31B, XY31A, and XX54A are control fibroblasts. The patient-derived fibroblasts are samples T1 and T2. GAPDH served as the reference gene for quantification of AR and FKBP5 mRNA (panels a and b). The expression of all samples was normalized to the control sample XX31B vehicle treatment. The bars represent mean ± SD of 3–5 independent samples. In (c), AR was immunoprecipitated with rabbit polyclonal α-AR17 and detected in western blotting with mouse monoclonal α-AR 441 recognizing AR amino acids 299–315 in the AR N-terminal domain. α-GAPDH was used to control the loading of input samples. The samples in the different blots are from the same experiment. The blots have been cropped; full-length blots are presented in Supplementary Figure S1.

Mentions: Both aberrant splicing events result in mRNA isoforms which code for 12 additional amino acids (NRIQLSFPLRSP) followed by a premature stop codon (PTC) after amino acid 816. Because of an intron downstream of the PTC, both aberrantly spliced mRNA isoforms are potential targets for the nonsense-mediated decay (NMD) pathway that would lead to the degradation of the AR mRNA7. Compared to LNCaP prostate cancer cells expressing biologically active levels of the AR, both control fibroblasts and patient samples expressed very low amounts of AR mRNA (≤10% of the LNCaP levels, Fig. 2a). This difference was also evident on the AR protein level (Fig. 2c). Additionally, even though the patient fibroblasts expressed overall similar amounts of the AR mRNA as the controls, quantification of the normally-spliced AR mRNA in the patient fibroblasts showed only approximately 10% level of expression as compared to the XX31B control fibroblasts (T1: 9.0–10.9% expression, T2: 11.6–12.9% expression) (Fig. 3). In accordance, immunoprecipitation with a polyclonal anti-AR antibody-coupled to western blotting with a monoclonal anti-AR antibody showed no signs of the AR protein in the patient samples as opposed to two of the control samples. The functionality of androgen signaling in the cells was also tested by examining the ability of the AR agonist R1881 to induce the expression of the AR-target gene FKBP5, but even in the control fibroblasts, the amount of the AR was not sufficient for any induction, whereas the induction was clearly detectable in the LNCaP cells (Fig. 2b).


Complete androgen insensitivity syndrome caused by a deep intronic pseudoexon-activating mutation in the androgen receptor gene
Expression of AR mRNA (a), androgen target gene FKBP5 (b), and AR protein (c) in fibroblasts and LNCaP prostatic cancer cells. The cells were split onto 6-well plates, and after 24 h, the medium was changed to steroid-depleted medium for 6 h. A half of the wells were treated with vehicle (0.1% ethanol) (−) and a half with 1 nM R1881 (+) for 18 h before immunoprecipitation or RNA extraction. Samples XX31B, XY31A, and XX54A are control fibroblasts. The patient-derived fibroblasts are samples T1 and T2. GAPDH served as the reference gene for quantification of AR and FKBP5 mRNA (panels a and b). The expression of all samples was normalized to the control sample XX31B vehicle treatment. The bars represent mean ± SD of 3–5 independent samples. In (c), AR was immunoprecipitated with rabbit polyclonal α-AR17 and detected in western blotting with mouse monoclonal α-AR 441 recognizing AR amino acids 299–315 in the AR N-terminal domain. α-GAPDH was used to control the loading of input samples. The samples in the different blots are from the same experiment. The blots have been cropped; full-length blots are presented in Supplementary Figure S1.
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Related In: Results  -  Collection

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f2: Expression of AR mRNA (a), androgen target gene FKBP5 (b), and AR protein (c) in fibroblasts and LNCaP prostatic cancer cells. The cells were split onto 6-well plates, and after 24 h, the medium was changed to steroid-depleted medium for 6 h. A half of the wells were treated with vehicle (0.1% ethanol) (−) and a half with 1 nM R1881 (+) for 18 h before immunoprecipitation or RNA extraction. Samples XX31B, XY31A, and XX54A are control fibroblasts. The patient-derived fibroblasts are samples T1 and T2. GAPDH served as the reference gene for quantification of AR and FKBP5 mRNA (panels a and b). The expression of all samples was normalized to the control sample XX31B vehicle treatment. The bars represent mean ± SD of 3–5 independent samples. In (c), AR was immunoprecipitated with rabbit polyclonal α-AR17 and detected in western blotting with mouse monoclonal α-AR 441 recognizing AR amino acids 299–315 in the AR N-terminal domain. α-GAPDH was used to control the loading of input samples. The samples in the different blots are from the same experiment. The blots have been cropped; full-length blots are presented in Supplementary Figure S1.
Mentions: Both aberrant splicing events result in mRNA isoforms which code for 12 additional amino acids (NRIQLSFPLRSP) followed by a premature stop codon (PTC) after amino acid 816. Because of an intron downstream of the PTC, both aberrantly spliced mRNA isoforms are potential targets for the nonsense-mediated decay (NMD) pathway that would lead to the degradation of the AR mRNA7. Compared to LNCaP prostate cancer cells expressing biologically active levels of the AR, both control fibroblasts and patient samples expressed very low amounts of AR mRNA (≤10% of the LNCaP levels, Fig. 2a). This difference was also evident on the AR protein level (Fig. 2c). Additionally, even though the patient fibroblasts expressed overall similar amounts of the AR mRNA as the controls, quantification of the normally-spliced AR mRNA in the patient fibroblasts showed only approximately 10% level of expression as compared to the XX31B control fibroblasts (T1: 9.0–10.9% expression, T2: 11.6–12.9% expression) (Fig. 3). In accordance, immunoprecipitation with a polyclonal anti-AR antibody-coupled to western blotting with a monoclonal anti-AR antibody showed no signs of the AR protein in the patient samples as opposed to two of the control samples. The functionality of androgen signaling in the cells was also tested by examining the ability of the AR agonist R1881 to induce the expression of the AR-target gene FKBP5, but even in the control fibroblasts, the amount of the AR was not sufficient for any induction, whereas the induction was clearly detectable in the LNCaP cells (Fig. 2b).

View Article: PubMed Central - PubMed

ABSTRACT

Mutations in the X-linked androgen receptor (AR) gene underlie complete androgen insensitivity syndrome (CAIS), the most common cause of 46,XY sex reversal. Molecular genetic diagnosis of CAIS, however, remains uncertain in patients who show normal coding region of AR. Here, we describe a novel mechanism of AR disruption leading to CAIS in two 46,XY sisters. We analyzed whole-genome sequencing data of the patients for pathogenic variants outside the AR coding region. Patient fibroblasts from the genital area were used for AR cDNA analysis and protein quantification. Analysis of the cDNA revealed aberrant splicing of the mRNA caused by a deep intronic mutation (c.2450-118A>G) in the intron 6 of AR. The mutation creates a de novo 5′ splice site and a putative exonic splicing enhancer motif, which leads to the preferential formation of two aberrantly spliced mRNAs (predicted to include a premature stop codon). Patient fibroblasts contained no detectable AR protein. Our results show that patients with CAIS and normal AR coding region need to be examined for deep intronic mutations that can lead to pseudoexon activation.

No MeSH data available.


Related in: MedlinePlus