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Contains fulltext : 197541.pdf (Publisher’s version ) (Closed access), Context: Inactivating mutations within the AR-gene are present in only around 40% of individuals with clinically and hormonally diagnosed androgen insensitivity syndrome (AIS). Previous studies revealed the existence of an AR-gene mutation negative group of AIS-individuals with compromised AR-function (AIS type II). Objective: To investigate if AIS type II can be due to epigenetic repression of AR-transcription. Design: Quantification of AR-mRNA and AR proximal promoter CpG-methylation levels in genital skin derived fibroblasts (GF) derived from AIS type II individuals and control individuals. Setting: University Hospital endocrine research laboratory. Patients: GF from control individuals (N=11) and AIS type II individuals (N=14). Intervention(s): None. Main Outcome Measure(s): Measurement of AR-mRNA and AR promoter CpG-methylation as well as activity of AR proximal promoter in vitro. Results: 57% of individuals with AIS type II (N=8) show a reduced AR-mRNA expression in their GF. A significant inverse correlation exists between AR-mRNA abundance and methylation at two consecutive CpGs within the proximal AR promoter. Methylation of a 158bp long region containing these CpGs is sufficient to severely reduce reporter gene expression. This region is bound by the Runt Related transcription factor1 (RUNX1). Ectopic expression of RUNX1 in HEK293T cells is able to inhibit reporter gene expression through this region. Conclusions: Aberrant CpGs methylation within the proximal AR promoter plays an important role in the control of AR-gene expression and may result in AIS type II. We suggest that transcriptional modifiers, like RUNX1, could play roles therein offering new perspectives for understanding androgen-mediated endocrine diseases.

A subset of patients with monogenic disorders lacks disease causing mutations in the protein coding region of the corresponding gene. Here we describe a recurrent germline mutation found in two unrelated patients with complete androgen insensitivity syndrome (CAIS) generating an upstream open reading frame (uORF) in the 5’ untranslated region (5’-UTR) of the androgen receptor (AR) gene. We show in patient derived primary genital skin fibroblasts as well as in cell-based reporter assays that this mutation severely impacts AR function by reducing AR protein levels without affecting AR mRNA levels. Importantly, the newly generated uORF translates into a polypeptide and the expression level of this polypeptide inversely correlates with protein translation from the primary ORF of the AR thereby providing a model for AR-5′UTR mediated translational repression. Our findings not only add a hitherto unrecognized genetic cause to complete androgen insensitivity but also underline the importance of 5′UTR mutations affecting uORFs for the pathogenesis of monogenic disorders in general.

The human androgen receptor is a member of the superfamily of steroid hormone receptors. Proper functioning of this protein is a prerequisite for normal male sexual differentiation and development. The cloning of the human androgen receptor cDNA and the elucidation of the genomic organization of the corresponding gene has enabled us to study androgen receptors in subjects with the clinical manifestation of androgen insensitivity and in a human prostate carcinoma cell line (LNCaP). Using PCR amplification, subcloning and sequencing of exons 2–8, we identified a G → T mutation in the androgen receptor gene of a subject with the complete form of androgen insensitivity, which inactivates the splice donor site at the exon 4/intron 4 boundary. This mutation causes the inactivation of a cryptic splice donor site in exon 4, which results in the deletion of 41 amino acids from the steroid binding domain. In two other independently arising cases we identified two different nucleotide alterations in codon 686 (GAC; aspartic acid) located in exon 4. One mutation (G → C) results in an aspartic acid → histidine substitution (with negligible androgen binding), whereas the other mutation (G → A) leads to an aspartic acid → asparagine substitution (normal androgen binding, but a rapidly dissociating androgen receptor complex). Sequence analysis of the androgen receptor in human LNCaP-cells (lymph node carcinoma of the prostate) revealed a point mutation (A → G) in codon 868 in exon 8 resulting in the substitution of threonine by alanine. This mutation is the cause of the altered steroid binding specificity of the LNCaP-cell androgen receptor. The functional consequences of the observed mutations with respect to protein expression, specific ligand binding and transcriptional activation, were established after transient expression of the mutant receptors in COS and HeLa cells. These findings illustrate that functional errors