Dagmar Struve

Complete androgen insensitivity caused by a splice donor site mutation in intron 2 of the human androgen receptor gene resulting in an exon 2-lacking transcript with premature stop-codon and reduced expression

Various mutations within the human androgen receptor gene have been documented to cause defective sexual differentiation in karyotypic male individuals. In this study, we report a previously undescribed point mutation at the donor splice-site of the second intron of the androgen receptor gene in a patient with a completely female phenotype. The sequence alteration was detected by single-strand-conformation-analysis-PCR and genomic sequencing. Applying competitive reverse transcribed PCR, cDNA sequencing and Western blotting, we could demonstrate considerable aberrations of structure and concentration of the transcript and its translation product in the patients fibroblasts from the genital region. (1) In the transcript, exon 1 and 3 are directly linked to each other, the complete second exon is skipped. The mRNA predictively suffers a codon frame-shift in exon 3 associated with a premature termination between codons 598 and 599, leading to a truncated androgen receptor protein lacking any in vivo function. (2) Steady-state concentration levels of transcript and protein are abnormally low. Our observations highlight the influence of exon-flanking intron sequences on proper expression and function of gene products.

A positive genotype-phenotype correlation in a large cohort of patients with Pseudohypoparathyroidism Type Ia and Pseudo-pseudohypoparathyroidism and 33 newly identified mutations in the GNAS gene.

Maternally inherited inactivating GNAS mutations are the most common cause of parathyroid hormone (PTH) resistance and Albright hereditary osteodystrophy (AHO) leading to pseudohypoparathyroidism type Ia (PHPIa) due to Gsα deficiency. Paternally inherited inactivating mutations lead to isolated AHO signs characterizing pseudo‐pseudohypoparathyroidism (PPHP). Mutations are distributed throughout the Gsα coding exons of GNAS and there is a lack of genotype–phenotype correlation. In this study, we sequenced exon 1–13 of GNAS in a large cohort of PHPIa‐ and PPHP patients and identified 58 different mutations in 88 patients and 27 relatives. Thirty‐three mutations including 15 missense mutations were newly discovered. Furthermore, we found three hot spots: a known hotspot (p.D190MfsX14), a second at codon 166 (p.R166C), and a third at the exon 5 acceptor splice site (c.435 + 1G>A), found in 15, 5, and 4 unrelated patients, respectively. Comparing the clinical features to the molecular genetic data, a significantly higher occurrence of subcutaneous calcifications in patients harboring truncating versus missense mutations was demonstrated. Thus, in the largest cohort of PHPIa patients described to date, we extend the spectrum of known GNAS mutations and hot spots and demonstrate for the first time a correlation between the genetic defects and the expression of a clinical AHO‐feature.

Expression of two functionally different androgen receptors in a patient with androgen insensitivity

Abstract Recently, we demonstrated a previously unknown high rate of de novo mutations of the androgen receptor (AR) gene in androgen insensitivity syndrome (AIS) with some resulting in somatic mosaicism of mutant and wild type AR alleles. However, data on the genotype-phenotype relationship in the latter patients are sparse. We present here a 46,XY newborn with ambiguous genitalia carrying a mosaic of an 866 GTG (Val) → ATG (Met) mutation with the wild type AR gene. This mutation has usually been associated with complete AIS. Accordingly, we found markedly impaired transactivation due to the mutant Met866 AR. Essential information arose from Scatchard analysis of methyltrienolone binding on cultured genital skin fibroblasts. We demonstrated for the first time the expression of two functionally different ARs (Kd1: 5.58 nM = mutant, Kd2: 0.06 nM = wild type) in one AIS individual. This finding not only represents an important confirmation for the presence of the somatic mosaicism in the patient, it also indicates the most likely molecular mechanism responsible for the unexpectedly strong virilization of the patient: Androgen action through the wild type AR expressed by part of the somatic cells. Conclusions The present case clearly demonstrates the molecular mechanism by which somatic mosaicism of the androgen receptor gene can modulate in vivo androgen action. It underlines the importance of particular notice on somatic mosaicism in all androgen insensitivity syndrome patients carrying de novo mutations of the androgen receptor gene.

In-vitro Characterization of Androgen Receptor Mutations Associated with Complete Androgen Insensitivity Syndrome Reveals Distinct Functional Deficits

Adequate androgen receptor (AR) function is crucial for male sex development and maintenance of secondary male characteristics. Mutations in the AR lead to androgen insensitivity syndrome (AIS) characterized by an end-organ resistance to androgens. The clinical appearance of individuals with 46,XY karyotype and an AR mutation varies widely from normal male to the ultimate completely female phenotype of complete androgen insensitivity syndrome (CAIS). We have analyzed the androgen receptor missense mutations P723S, P904S, and H917R, clinically associated with CAIS, which were described to have a normal maximum androgen binding (Bmax) but elevated equilibrium dissociation constants (Kd’s) and compared their properties with the F916X deletion mutant, leading to the loss of the last four amino acids of the AR. Functional analysis allowed a quantitative and qualitative discrimination of these mutants in transactivation, amino-terminal/carboxy-terminal (N/C)-interaction, and coactivation capacity, varying widely with each distinct mutation. We conclude that mutations in the AR have to be characterized meticulously, not only to prove any quantitative functional deficit as a proof of consequence, but also to gain knowledge on qualitative functional properties. This is necessary as a possible link to genotype-phenotype correlation in AIS, but also with respect to medical decision making in CAIS.

RWDD1 interacts with the ligand binding domain of the androgen receptor and acts as a coactivator of androgen-dependent transactivation.

During embryogenesis, the development of the male genital is dependent on androgens. Their actions are mediated by the androgen receptor (AR), which functions as a transcription factor. To identify AR coregulators that support AR action during the critical time window of androgen-dependent development in the genital tubercle of male mice, we performed yeast two-hybrid screenings with cDNA libraries of genital tubercles from male mouse embryos using human AR as bait. RWD domain containing 1 (RWDD1) was identified as an AR-interacting protein from three independent libraries of the embryonic days E15, E16 and E17. The interaction between the AR and RWDD1 was confirmed in vitro and in vivo and the ligand binding domain of the AR was shown to be sufficient to mediate the interaction. RWDD1 enhanced AR-dependent transactivation in reporter assays with promoters of different complexity and in different cell lines. These results suggest that RWDD1 functions as a coactivator of androgen-dependent transcription.

Preserved Fertility in a Patient with Gynecomastia Associated with the p.Pro695Ser Mutation in the Androgen Receptor

The androgen insensitivity syndrome (AIS) is described as a dysfunction of the androgen receptor (AR) in 46,XY individuals, which can be associated with mutations in the AR gene or can be due to unknown mechanisms. Different mutations in AIS generally cause variable phenotypes that range from a complete hormone resistance to a mild form usually associated with male infertility. The purpose of this study was to search for mutations in the AR gene in a fertile man with gynecomastia and to evaluate the influence of the mutation on the AR transactivation ability. Sequencing of the AR gene revealed the p.Pro695Ser mutation. It is located within the AR ligand-binding domain. Bioinformatics analysis indicated a deleterious role, which was verified after testing transactivation activity and N-/C-terminal (N/C) interaction by in vitro expression of a reporter gene and 2-hybrid assays. p.Pro695Ser showed low levels of both transactivation activity and N/C interaction at low dihydrotestosterone (DHT) conditions. As the ligand concentration increased, both transactivation activity and N/C interaction also increased and reached normal levels. Therefore, this study provides functional insights for the p.Pro695Ser mutation described here for the first time in a patient with mild AIS. The expression profile of p.Pro695Ser not only correlates to the patients phenotype, but also suggests that a high-dose DHT therapy may overcome the functional deficit of the mutant AR.

Functional characterization of five NR5A1 gene mutations found in patients with 46,XY disorders of sex development

Steroidogenic factor‐1 (SF1), encoded by the NR5A1 gene, is a key regulator of steroidogenesis and reproductive development. NR5A1 mutations described in 46,XY patients with disorders of sex development (DSD) can be associated with a range of conditions of phenotypes; however, the genotype–phenotype correlation remains elusive in many cases. In the present study, we describe the impact of five NR5A1 variants (three novel: p.Arg39Cys, p.Ser32Asn, and p.Lys396Argfs*34; and two previously described: p.Cys65Tyr and p.Cys247*) on protein function, identified in seven patients with 46,XY DSD. In vitro functional analyses demonstrate that NR5A1 mutations impair protein functions and result in the DSD phenotype observed in our patients. Missense mutations in the DNA binding domain and the frameshift mutation p.Lys396Argfs*34 lead to both, markedly affected transactivation assays, and loss of DNA binding, whereas the mutation p.Cys247* retained partial transactivation capacity and the ability to bind a consensus SF1 responsive element. SF1 acts in a dose‐dependent manner and regulates a cascade of genes involved in the sex determination and steroidogenesis, but in most cases reported so far, still lead to a sufficient adrenal steroidogenesis and function, just like in our cases, in which heterozygous mutations are associated to 46,XY DSD with intact adrenal steroid biosynthesis.

Functional Impact of Novel Androgen Receptor Mutations on the Clinical Manifestation of Androgen Insensitivity Syndrome

Androgens are responsible for the development and maintenance of male sex characteristics. Dysfunctions in androgen action due to mutations in the androgen receptor gene (AR) can lead to androgen insensitivity syndrome (AIS) that can be classified as mild (MAIS), partial (PAIS), or complete (CAIS). We have analyzed functional effects of p.Ser760Thr, p.Leu831Phe, p.Ile899Phe, p.Leu769Val, and p.Pro905Arg mutations and the combination p.Gln799Glu + p.Cys807Phe that were identified in patients with PAIS or CAIS. The p.Leu769Val and p.Pro905Arg mutations showed complete disruption of AR action under physiological hormone concentrations; however, they differed in high DHT concentrations especially in the N/C terminal interaction assay. Mutations p.Ser760Thr, p.Leu831Phe, p.Ile899Phe presented transactivation activities higher than 20% of the wild type in physiological hormone concentrations and increased with higher DHT concentrations. However, each one showed a different profile in the N/C interaction assay. When p.Gln799Glu and p.Cys807Phe were analyzed in combination, transactivation activities <10% in physiologic hormone conditions indicated an association with a CAIS phenotype. We conclude that the functional analysis elucidated the role of mutant ARs, giving clues for the molecular mechanisms associated with different clinical AIS manifestations. Differences in hormone-dependent profiles may provide a basis for the response to treatment in each particular case.

Contents Vol. 8, 2014

Karin Schmid (address as for M. Schmid) E-mail: karin.schmid@uni-wuerzburg.de Peter Koopman Professor of Developmental Biology Institute for Molecular Bioscience The University of Queensland AU–Brisbane, Qld. 4072 (Australia) Tel. (+61) 7 3346 2059; Fax. (+61) 7 3346 2101 E-mail p.koopman@imb.uq.edu.au Manfred Schartl Institute of Physiological Chemistry I University of Würzburg Biozentrum, Am Hubland D–97074 Würzburg (Germany) Tel. (+49) 931 318 4148; Fax (+49) 931 318 4150 E-mail: phch1@biozentrum.uni-wuerzburg.de