A unique exonic splicing mutation in the human androgen receptor gene indicates a physiologic relevance of regular androgen receptor transcript variants.

In a patient with partial androgen insensitivity syndrome (AIS), we identified a single inherited presumably silent nucleotide variation (AGC -> AGT) in exon 8 (codon 888) of the AR gene. However, in the patient's genital skin fibroblasts, a considerably shortened transcript of 5.5 kb (normal: 10.5 kb) was detected, which misses a part of exon 8 and a prominent portion of the 3'-untranslated region. The translation product includes eight missense amino acids from codon 886 onward followed by a premature stop codon. As shown by in vitro expression analysis, the mutant protein lacks any residual function. However, reverse transcribed PCRs and sequence data indicate the existence of two additional splicing variants of 6.4 kb and 7.8-kb length both in patient and normal control genital skin fibroblasts. These splicing variants comprise the complete coding region but a shortened 3'-untranslated region. Thus, a distinct alternative pre-messenger RNA-processing event leading to two additional transcripts occurs generally in genital skin fibroblasts. In addition, this process partially prevents aberrant splicing in the patient and produces a small fraction of normal, functionally intact AR-protein that could explain the partial masculinization in this patient. This first report of an exonic splicing mutation in the AR-gene indicates a physiologic relevance of the regular AR-messenger RNA variants with shortened 3'-untranslated regions and their functional translation products in human genital development.

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 patient's 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.

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

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. 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.