Intrinsic androgen-dependent gene expression patterns revealed by comparison of genital fibroblasts from normal males and individuals with complete and partial androgen insensitivity syndrome

<p>Abstract</p> <p>Background</p> <p>To better understand the molecular programs of normal and abnormal genital development, clear-cut definition of androgen-dependent gene expression patterns, without the influence of genotype (46, XX vs. 46, XY), is warranted. Previously, we have identified global gene expression profiles in genital-derived fibroblasts that differ between 46, XY males and 46, XY females with complete androgen insensitivity syndrome (CAIS) due to inactivating mutations of the androgen receptor (AR). While these differences could be due to cell autonomous changes in gene expression induced by androgen programming, recent work suggests they could also be influenced by the location from which the fibroblasts were harvested (topology). To minimize the influence of topology, we compared gene expression patterns of fibroblasts derived from identical urogenital anlagen: the scrotum in normally virilized 46, XY males and the labia majora from completely feminized 46, XY individuals with CAIS.</p> <p>Results</p> <p>612 transcripts representing 440 unique genes differed significantly in expression levels between scrotum and CAIS labia majora, suggesting the effects of androgen programming. While some genes coincided with those we had identified previously (TBX3, IGFBP5, EGFR, CSPG2), a significant number did not, implying that topology had influenced gene expression in our previous experiments. Supervised clustering of gene expression data derived from a large set of fibroblast cultures from individuals with partial AIS revealed that the new, topology controlled data set better classified the specimens.</p> <p>Conclusion</p> <p>Inactivating mutations of the AR, in themselves, appear to induce lasting changes in gene expression in cultured fibroblasts, independent of topology and genotype. Genes identified are likely to be relevant candidates to decipher androgen-dependent normal and abnormal genital development.</p

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

Characterization of the Testicular Abnormality in 5a-Reductase Deficiency

The testes of five phenotypic women (from four families) with 5ª-reductase deficiency were studied. In one of the patients, the enzyme deficiency was similar in the testis and epididymis and in fibroblasts cultured from the labia majora. In testes from four of the patients, the concentrations of the 55ªreduced steroids dihydrotestosterone and 3ª-androstanediol were less than 10% of those in normal subjects. We conclude that the testis is involved in 55ª-reductase deficiency. Impaired spermatogenesis was evident in testicular biopsies from all five subjects, and in two, sperm production, as estimated in testicular homogenates, was less than 10% of normal. The extent to which spermatogenic arrest is due to 55ª-reductase deficiency or testicular maldescent is not clear

Intrinsic androgen-dependent gene expression patterns revealed by comparison of genital fibroblasts from normal males and individuals with complete and partial androgen insensitivity syndrome-1

<p><b>Copyright information:</b></p><p>Taken from "Intrinsic androgen-dependent gene expression patterns revealed by comparison of genital fibroblasts from normal males and individuals with complete and partial androgen insensitivity syndrome"</p><p>http://www.biomedcentral.com/1471-2164/8/376</p><p>BMC Genomics 2007;8():376-376.</p><p>Published online 18 Oct 2007</p><p>PMCID:PMC2212662.</p><p></p>interpretable data across the experiments whose expression levels were at least 2-fold different from the mean expression across all samples in at least 5 microarrays. (A) The cluster dendrogram demonstrating the degree of relatedness (Pearson correlation) between the expression patterns of the 259 genes in the cultured fibroblast samples. The length of the arms of the dendrogram reflects the degree of correlation between the samples. Samples are color coded to reflect the localization of the biopsy and the degree of external genital virilization according to a grading scheme developed by Sinnecker et al. [10]. The grey bar below indicates whether a sample was derived from dataset 1, 2 or 3. ''L.n.d.'' signifies that the biopsy localization was not accurately documented. Italics indicate a sample with a 46, XX karyotype. (B) Schematic depiction of the external genitalia phenotype of the cases from which the fibroblast cultures were derived using color coding that corresponds to the degree of genital ambiguity and the location of the biopsy. Color coding corresponds to the bar below the dendrogram in (A). (C) Cluster of AR-dependent transcripts that are highly expressed in the left ''male'' major branch of the cluster that are expressed at significantly lower levels in the ''female'' branch of the cluster on the right. TBX3, previously reported in ulnar mammary syndrome and IGF2, previously reported as being down-regulated in CAIS [20] are shown in this cluster. (D) Cluster of AR-dependent transcripts that are expressed at significantly lower levels in the lefthand ''male'' branch of the cluster and at higher levels in the righthand ''female'' branch. These include many extracellular matrix genes such as proteoglycan testican, versican, and fibrillin 1. Detailed data on figure 2 is available in additional files , , ,

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

Intrinsic androgen-dependent gene expression patterns revealed by comparison of genital fibroblasts from normal males and individuals with complete and partial androgen insensitivity syndrome-4

<p><b>Copyright information:</b></p><p>Taken from "Intrinsic androgen-dependent gene expression patterns revealed by comparison of genital fibroblasts from normal males and individuals with complete and partial androgen insensitivity syndrome"</p><p>http://www.biomedcentral.com/1471-2164/8/376</p><p>BMC Genomics 2007;8():376-376.</p><p>Published online 18 Oct 2007</p><p>PMCID:PMC2212662.</p><p></p>ual transcripts are grouped by hierarchical cluster analysis and are displayed in rows while experiments are represented in columns. Expression values per gene are centered by the mean logred/green normalized ratio. Increasing red intensity corresponds to higher relative transcript levels compared to the mean expression level across all 15 array experiments. Increasing green intensity corresponds to relatively decreased transcript levels compared to the mean. On the right side, examples of individual genes (gene symbols according to S.O.U.R.C.E. []) discussed in the paper or falling into the biological processes and cellular pathways detected by PANTHER are displayed. Detailed data on figure 1 is available in additional files , , ,

Intrinsic androgen-dependent gene expression patterns revealed by comparison of genital fibroblasts from normal males and individuals with complete and partial androgen insensitivity syndrome-2

<p><b>Copyright information:</b></p><p>Taken from "Intrinsic androgen-dependent gene expression patterns revealed by comparison of genital fibroblasts from normal males and individuals with complete and partial androgen insensitivity syndrome"</p><p>http://www.biomedcentral.com/1471-2164/8/376</p><p>BMC Genomics 2007;8():376-376.</p><p>Published online 18 Oct 2007</p><p>PMCID:PMC2212662.</p><p></p>on (B). In each of the two clusters A and B, the microarray experiments used to generate the underlying list of significant genes by SAM were removed before clustering resulting 57 remaining experiments in (A) and 58 in (B), respectively. The color code is the same as in Fig. 2. Based on the previous gene set, several highly virilized AIS 2 patients and normal scrotal fibroblasts were incorrectly classified female

Intrinsic androgen-dependent gene expression patterns revealed by comparison of genital fibroblasts from normal males and individuals with complete and partial androgen insensitivity syndrome-3

<p><b>Copyright information:</b></p><p>Taken from "Intrinsic androgen-dependent gene expression patterns revealed by comparison of genital fibroblasts from normal males and individuals with complete and partial androgen insensitivity syndrome"</p><p>http://www.biomedcentral.com/1471-2164/8/376</p><p>BMC Genomics 2007;8():376-376.</p><p>Published online 18 Oct 2007</p><p>PMCID:PMC2212662.</p><p></p>normal scrotal cell lines. Semi-quantitative RT-PCR was performed on four samples of normal male scrotal fibroblasts (S4, S5, S8, S9), as well as on four samples of labia majora derived from CAIS patients (ARD402, ARD411, ARD682, ARD1097). Differences in expression levels between different cell lines were calculated according to the ΔΔ-CT method [19]. The y-axis represents the ratios of expression levels of CAIS labia majora fibroblasts divided by those from scrotal fibroblasts