A recurrent germline mutation in the 5’UTR of the androgen receptor causes complete androgen insensitivity by activating aberrant uORF translation

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

A Quantitative Proteomics Tool To Identify DNA–Protein Interactions in Primary Cells or Blood

Interactions between transcription factors and genomic DNA, and in particular their impact on disease and cell fate, have been extensively studied on a global level using techniques based on next-generation sequencing. These approaches, however, do not allow an unbiased study of protein complexes that bind to certain DNA sequences. DNA pulldowns from crude lysates combined with quantitative mass spectrometry were recently introduced to close this gap. Established protocols, however, are restricted to cell lines because they are based on metabolic labeling or require large amounts of material. We introduce a high-throughput-compatible DNA pulldown that combines on-bead digestion with direct dimethyl labeling or label-free protein quantification. We demonstrate that our method can efficiently identify transcription factors binding to their consensus DNA motifs in extracts from primary foreskin fibroblasts and peripheral blood mononuclear cells (PBMCs) freshly isolated from human donors. Nuclear proteomes with absolute quantification of nearly 7000 proteins in K562 cells and PBMCs clearly link differential interactions to differences in protein abundance, hence stressing the importance of selecting relevant cell extracts for any interaction in question. As shown for rs6904029, a SNP highly associated with chronic lymphocytic leukemia, our approach can provide invaluable functional data, for example, through integration with GWAS

Epigenetic Repression of Androgen Receptor Transcription in Mutation-Negative Androgen Insensitivity Syndrome (AIS Type II)

Context: Inactivating mutations within the AR gene are present in only ~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 patients with AIS who have compromised androgen receptor (AR) function (AIS type II). Objective: To investigate whether 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 (GFs) derived from patients with AIS type II and control individuals. Setting: University hospital endocrine research laboratory. Patients: GFs from control individuals (n = 11) and patients with AIS type II (n = 14). Main Outcome Measure(s): Measurement of AR mRNA and AR promoter CpG methylation as well as activity of AR proximal promoter in vitro. Results: Fifty-seven percent of individuals with AIS type II (n = 8) showed a reduced AR mRNA expression in their GFs. A significant inverse correlation was shown between AR mRNA abundance and methylation at two consecutive CpGs within the proximal AR promoter. Methylation of a 158-bp-long region containing these CpGs was sufficient to severely reduce reporter gene expression. This region was bound by the runt related transcription factor 1 (RUNX1). Ectopic expression of RUNX1 in HEK293T cells was 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, such as RUNX1, could play roles therein offering new perspectives for understanding androgen-mediated endocrine diseases

Epigenetic Repression of Androgen Receptor Transcription in Mutation-Negative Androgen Insensitivity Syndrome (AIS Type II)

Context: Inactivating mutations within the AR gene are present in only ~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 patients with AIS who have compromised androgen receptor (AR) function (AIS type II). Objective: To investigate whether 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 (GFs) derived from patients with AIS type II and control individuals. Setting: University hospital endocrine research laboratory. Patients: GFs from control individuals (n = 11) and patients with AIS type II (n = 14). Main Outcome Measure(s): Measurement of AR mRNA and AR promoter CpG methylation as well as activity of AR proximal promoter in vitro. Results: Fifty-seven percent of individuals with AIS type II (n = 8) showed a reduced AR mRNA expression in their GFs. A significant inverse correlation was shown between AR mRNA abundance and methylation at two consecutive CpGs within the proximal AR promoter. Methylation of a 158-bp-long region containing these CpGs was sufficient to severely reduce reporter gene expression. This region was bound by the runt related transcription factor 1 (RUNX1). Ectopic expression of RUNX1 in HEK293T cells was 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, such as RUNX1, could play roles therein offering new perspectives for understanding androgen-mediated endocrine diseases

Next generation sequencing of the AR in the two index patients.

<p>A) Schematic representation of the <i>AR</i>-5′UTRs and CDS. The position of the c.-547C>T mutation and the uORF are indicated. B) A Haloplex library spanning the coding region, introns, UTRs and up and downstream sequences of the AR genomic locus was prepared from DNA of the index patients′ GF and sequenced on a MiSeq benchtop sequencer. Analysis for single nucleotide polymorphisms (SNP) and small insertion deletions (indels) was performed by the SureCall software (Agilent). Indicated is the mutation found in the 5′UTR of the <i>AR</i>. A frequency of 1 corresponds to 100% of the reads. The depth indicates the number of reads covering the indicated genomic position. C) Sanger sequencing of a male control and the index patients 1 and 2. The sequences are visualized as reverse complement strand using the Chromas Lite software and show the c.-547C>T mutation in both index patients but not in the male control. D) Sanger sequencing of blood derived DNA from both index patients as well as from the mothers of the index patients. The sequences are visualized as reverse complement strand using the Chromas Lite software and show the c.-547C>T mutation in both index patients and in the patients′ mothers in a heterozygous constellation.</p

Additional data on index patient 2.

<p>Additional data on index patient 2.</p

The c.-547C>T mutation in the <i>AR</i>-5′UTR reduces translation and AR activity <i>in vitro</i>.

<p>HEK293 cells were transfected with either empty vector, <i>AR</i>5′UTRwt-<i>GFP</i> or <i>AR</i>5′UTRmut-<i>GFP</i>. After 72h of transfection RNA and protein was isolated. A) Q-RT-PCR analysis of GFP mRNA. There is no significant difference between <i>GFP</i> mRNA levels of the <i>AR</i>5′UTRwt-<i>GFP</i> and the <i>AR</i>5′UTRmut-<i>GFP</i> construct (p = 0.57). <i>GFP</i> mRNA levels were normalized to the neomycin resistance (neo) expression of the vector. Experiments were performed in triplicate. B) GFP protein analysis. Cells transfected with the <i>AR</i>5′UTRmut-<i>GFP</i> construct show a reduced expression of the GFP protein compared to the wt-construct. GAPDH measurement served as loading control. The experiment was done in triplicate. One representative blot is displayed. C) FACS analysis of <i>AR</i>5′UTR-<i>GFP</i> transfected cells. FACS analysis was performed equally 72h after transfection. Cells transfected with <i>AR</i>5′UTRmut-<i>GFP</i> show less fluorescent intensity. D) Transcriptional activity of <i>AR</i>5′UTRwt-<i>AR and AR</i>5′UTRmut-<i>AR</i>. HEK293 cells were transfected with either empty vector, <i>AR</i>5′UTRwt-<i>AR</i> or <i>AR</i>5′UTRmut-<i>AR</i>. After 48h of transfection luciferase activity was measured. AR induced luciferase expression is significantly lower in <i>AR</i>5′UTRmut-<i>AR</i> transfected cells in respect to <i>AR</i>5′UTRwt-<i>AR</i> transfected cells (p***<0.001).</p

Additional data on index patient 1.

<p>Additional data on index patient 1.</p