Pedigree analysis of <i>CYP17A1</i> gene.

<p>The patient is found to be a compound heterozygous for mutations of <i>CYP17A1</i>. The first mutation was identified as IVS1 +2T>C and the second mutation p.R358X (CGA→TGA). The first intron mutation is originated from her mother and p.R358X from her father, based on the analysis of her uncle's genomic DNA. Proband's brother is also heterozygous for the affected maternal allele.</p

Heterologous expression and mRNA analysis using minigene and full-length <i>CYP17A1</i> gene, wild-type and mutation IVS1 +2T>C.

<p>A) RT-PCR analysis shows only the correct 311 bp amplicon when wild-type <i>CYP17A1</i> minigene or full-length gene is expressed in COS-7 or HEK293 cells. In contrast, the IVS1 +2T>C mutant <i>CYP17A1</i> gene affords primarily a ∼400 bp amplicon, best demonstrated in COS-7 cells, as well as lesser amounts of the normal 311 bp amplicon and larger molecular species. B) Cartoon demonstrating size and sequence of aberrantly spliced transcripts obtained from transfected HEK293 cells expressing the IVS1 +2T>C <i>CYP17A1</i> mutation, obtained after TOPO TA subcloning and sequencing. The asterisks indicate position of in-frame termination codons. C) Cartoon demonstrating splice sites used to generate transcripts obtained from transfected HEK293 cells expressing the IVS1 +2T>C <i>CYP17A1</i> mutation. Figure length is not drawn to scale. D) Western blot analysis of protein obtained from transiently transfected HEK293 cells expressing the wild type (W) and IVS1 +2T>C mutation (Mu) full-length <i>CYP17A1</i> gene. Decreased CYP17A1 protein expression was noted for the IVS1 +2T>C mutant full-length <i>CYP17A1</i> gene compared to the wild-type gene.</p

Patient's adrenal steroid metabolites.

<p>ACTH, Adrenocorticotropic hormone; DHEA, dehydroepiandrosterone.</p><p>SI units were given in parentheses. Asterisk (*) indicates analytes assayed by LC-MS/MS.</p

RT and semi-nested PCR analysis of <i>CYP17A1</i> mRNA from the patient's LCL.

<p>A) Amplicons were derived from RT-PCR spanning exons 1 and 2 (lane 1, 311 bp band), exons 1 and 3 (lane 2, 644 bp band), or exons 1 and 6 (lane 3, 1191 bp band). The PCR primers used are indicated at bottom of the figure panel; N.S. indicates a non-specific PCR product. The 1191bp PCR product spans from exon 1 to exon 6, with primer e designed just 3′ to the p.R358X position. B) Direct sequencing (reverse strand) analysis of the 1191bp band showed only the p.R358X mutation without wild type sequence.</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

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

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

Additional data on index patient 1.

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