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

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

The uORF is translated into a polypeptide.

<p>Expression analysis of the HIS-tagged uORF. HEK293 cells were transfected with either <i>AR</i>5′UTRwtHIS-<i>GFP</i> or <i>AR</i>5′UTRmutHIS-<i>GFP</i>. After 48h of transfection, RNA and protein was isolated. A) GFP protein analysis. Cells transfected with the <i>AR</i>5′UTRmutHIS-<i>GFP</i> construct show a strong peptide expression and highly reduced GFP protein levels. GAPDH measurement served as loading control. The experiment was done in triplicate. One representative blot is displayed. B) Q-RT-PCR analysis of <i>GFP</i> mRNA. There is no significant difference in the <i>GFP</i> mRNA levels between the <i>AR</i>5′UTRwtHIS-<i>GFP</i> and the <i>AR</i>5′UTRmutHIS-<i>GFP</i> construct (p = 0.64). <i>GFP</i> mRNA levels were normalized to the neomycin resistance expression of the vector. Experiments were performed in triplicate. C) Model for impaired AR-translation. In the wild type (wt) situation the 40S ribosomal subunit scans the 5′UTR for the translational start codon of the AR coding sequence (CDS). The majority of ribosomal complex will form at the canonical AR-start codon and protein translation starts. In the mutant (mut) 5′UTR a full ribosomal complex is loaded at the start of the uORF generated though the c.-5467C>T mutation creating a polypeptide of 63 amino acids. Ribosome stalling and/or dissociation of the 60S subunit at the end of the uORF would cause a poor re-initiation at the AR pORF. Internal ribosomal binding may lead to re-initiation at the next available ATG within a Kozak sequence to produce a shorter (75 kD) AR protein.</p

Additional data on index patient 1.

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

Functional and expression analysis of the AR <i>in vivo</i> and <i>in vitro</i>.

<p>A) <i>AR</i> mRNA accumulation in GF. Total RNA was extracted from GF of a male control, a CAIS patient with a documented frameshift mutation in the <i>AR</i> gene and index patient 1. Means and standard deviations of three independent experiments are shown. B) DHT-dependent AR protein expression in GF. Whole cell protein lysates were extracted from GF of the male control, the index patient and the CAIS patient with a documented frameshift mutation and treated with 10nM DHT or ethanol, respectively. From all samples, 35 μg of protein were loaded, additionally 10 μg were loaded for the control sample to avoid overexposition. Immunoblot analysis shows a markedly reduced amount of the 112 kD full-length AR protein in the index patient and an increased detection of a shorter 75kD fragment. DHT treatment stabilizes full-length 112kD AR protein while the shorter 75kD fragment remains of similar intensity as compared to untreated GF. The corresponding bands are indicated by an arrow. An unspecific band is denoted by a star. GF from the CAIS patient served as negative control. Actin measurement served as loading control. C) DHT-dependent ectopic AR protein expression in PC3 cells. Cells were either treated with 10nM DHT or ethanol. 5′UTRmut-<i>AR</i> transfected cells show reduced AR protein expression compared to 5′UTRwt-<i>AR</i> transfected cells. AR-121 transfected cells express an AR-fragment of lower molecular weight. Actin measurement served as loading control. D) DHT-dependent activation of the AR target gene <i>APOD</i> is compromised in the index patient. AR activity was measured through the activation of the endogenous AR target gene <i>APOD</i>. This revealed a mean 3.4 fold activation of <i>APOD</i> in response to DHT stimulation in three male foreskin control derived cell lines. GF of the index patient, like GF of the CAIS patient with known mutation show a highly significant loss of <i>APOD</i> induction as compared to the male controls (p***<0.001). Means and standard deviations of three independent experiments are shown. p-values are calculated by a t-test. E) DHT-dependent activation of the AR target gene <i>PPAP2B</i> is compromised in the index patient. AR activity was measured through the activation of the endogenous AR target gene <i>PPAP2B</i>. This revealed a mean 1.54 fold activation of <i>PPAP2B</i> in response to DHT stimulation in three male foreskin control derived cell lines. GF of the index patient, like GF of the CAIS patient with known mutation show a significant loss of <i>PPAP2B</i> induction as compared to the male controls (p**<0.01). Means and standard deviations of at least three independent experiments are shown. p-values are calculated by a t-test.</p

Identification of an AR mutation-negative class of androgen insensitivity by determining endogenous AR activity

Context: Only approximately 85%of patients with a clinical diagnosis complete androgen insensitivity syndrome and less than 30%with partial androgen insensitivity syndrome can be explained by inactivating mutations in the androgen receptor (AR) gene. Objective: The objective of the study was to clarify this discrepancy by in vitro determination of AR transcriptional activity in individuals with disorders of sex development (DSD) and male controls. Design: Quantification of DHT-dependent transcriptional induction of the AR target gene apolipoprotein D (APOD) in cultured genital fibroblasts (GFs) (APOD assay) and next-generation sequencing of the complete coding and noncoding AR locus. Setting: The study was conducted at a university hospital endocrine research laboratory. Patients: GFs from 169 individuals were studied encompassing control males (n=68), molecular defined DSD other than androgen insensitivity syndrome (AIS; n = 18), AR mutation-positive AIS (n = 37), and previously undiagnosed DSD including patients with a clinical suspicion of AIS (n=46). Intervention(s): There were no interventions. Main Outcome Measure(s): DHT-dependent APOD expression in cultured GF and AR mutation status in 169 individuals was measured. Results:TheAPODassay clearly separated control individuals (healthy malesandmolecular defined DSD patients other than AIS) from genetically proven AIS (cutoff<2.3-foldAPOD-induction;100%sensitivity, 93.3%specificity, P < .0001). Of 46 DSD individuals with no AR mutation, 17 (37%) fell below the cutoff, indicating disrupted androgen signaling. Conclusions:ARmutation-positive AIS canbereliably identified by theAPODassay. Its combination with next-generation sequencing of the AR locus uncovered an AR mutation-negative, new class of androgen resistance, which we propose to name AIS type II. Our data support the existence of cellular components outside the AR affecting androgen signaling during sexual differentiation with high clinical relevance