Hans Udo Schweikert

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

BackgroundTo 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.Results612 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.ConclusionInactivating 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.

Testosterone Metabolism of Fibroblasts Grown From Prostatic Carcinoma, Benign Prostatic Hyperplasia and Skin Fibroblasts

The metabolism of [1,2,6,7-3H]testosterone was assessed in fibroblast monolayers derived from tissue of 5 prostates with benign hyperplasia (BPH), 4 prostates with carcinoma (PC), and 3 biopsy samples of skin, 2 nongenital skin (NG) and 1 genital skin. The following metabolites could be identified: androstanedione androstenedione, dihydrotestosterone, androsterone, epiandrosterone, androstane-3 alpha, 17 beta-diol and androstane-3 beta, 17 beta-diol. Testosterone was metabolized much more rapidly in fibroblasts originating from prostatic tissue than in fibroblasts derived from NG. A significantly higher formation of 5 alpha-androstanes and 3 alpha-hydroxysteroids could be observed in fibroblasts from BPH as compared to PC. 17-ketosteroid formation exceeded 5 alpha-androstane formation in BPH, whereas 5 alpha-reduction was the predominant pathway in fibroblasts grown from PC and NG. Since testosterone metabolism in fibroblasts of prostatic origin therefore resembles in many aspects that in whole prostatic tissue, fibroblasts grown from prostatic tissues might be a valuable tool for further investigation of the pathogenesis of human BPH and PC.

Androgen receptor isoforms AR-A and AR-B display functional differences in cultured human bone cells and genital skin fibroblasts

Two isoforms of the androgen receptor (AR-A and AR-B), differing by a lack of the first 187 amino acids in the NH2-terminal transactivation domain of AR-A, are expressed in connective tissue and bone. Transient transfections of normal human osteoblastic cells (HOB) and of genital skin fibroblasts defective in AR (GSF-540) were utilized to compare the functional properties of AR isoforms in mesenchymal tissues. Overexpression of AR-B or AR-A did not significantly affect type I collagen secretion. However, overexpression of AR-B (but not AR-A) restored androgen-dependent DNA synthesis in AR-defective fibroblasts and increased DHT-mediated DNA synthesis three-fold in osteoblastic cells. Overexpression of AR-A did not affect DHT action but reduced DHT-dependent DNA synthesis when transfected together with AR-B. The need for an NH2-terminal sequence of the AR for complete receptor function was demonstrated using electrophoretic mobility shift assay. A peptide coding for the amino terminus of the complete AR was able to decrease the binding affinity of AR-B and increase the binding affinity of AR-A to the androgen response element. Our results suggest that AR-A lacks the ability to stimulate cell proliferation possibly due to reduced binding of AR co-activating proteins to the truncated N-terminal transactivation domain rather than due to impaired stability of the AR-A isoform.

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 and functional characterization of the androgen receptor (AR) gene in patients with androgen insensitivity syndrome (AIS) and controls

Androgen insensitivity syndrome (AIS) is characterized by a partial to complete lack of genital virilization in genetically male individuals. It is classically caused by inactivating mutations in the coding region of the X-chromosomal androgen receptor (AR) gene. However, up to two-third of the patients with a clinically established diagnosis of AIS lack a detectable molecular cause to date. As conventional sequencing is restricted to the AR coding exons, we set up a next generation sequencing (NGS) approach of the entire AR-gene locus for a comprehensive AR mutation analysis in patients with AIS. To this purpose, DNA was extracted from cultured genital skin fibroblasts (GSF = scrotum, foreskin, labia) of 80 patients with known and presumed AIS, two patients with 17sHSDIII deficiency, six patients with 5alpha-reductase deficiency and 15 control males. Patients were suspected to have AIS based on clinical findings, pathological androgen binding, reduced AR expression in GSF or a combination of these. The AR-sequencing library was produced using a capture-based method (Haloplex; Agilent). The target sequence included the coding region, the UTRs, 90% of the intron sequences as well as a 9kb upstream and 5kb downstream sequence. Sequencing was performed on a Miseq benchtop sequencer (Illumina). Alignment to the hg19 reference genome and single nucleotide polymorphism (SNP) calling was performed by the MiSeq-Reporter software (Illumina). Targeted NGS confirmed AR mutations in all patients with mutations previously identified by Sanger sequencing. Additional SNPs were detected in and outside the AR-coding region. In order to understand the functional impact of the SNPs, they were further tested for the ability of the endogenous AR to induce transcription of the AR target gene Apolipoprotein D (APOD-Assay). In conclusion we show that NGS is a valid method for AR-sequencing in presumed AIS and allows in combination with the APOD-Assay a refined classification of AIS.

Endokrinologische Untersuchungen beim Reifenstein-Syndrom

Beim Reifenstein-Syndrom [1] handelt es sich um eine Form des familiaren, inkompletten mannlichen Pseudohermaphroditismus, die auf Grund neuerer Untersuchungen durch eine SubPartielle Resistenz androgener Zielorgane gegenuber der Wirkung von Testosteron verursacht wird [2, 3]. Es ist jedoch moglich, das andere Faktoren fur die Pathogenese ebenfalls von Bedeutung sind [4].