The role of DHEA sulfation in childhood androgen excess

Overproduction of male hormones can result in precocious pseudo-puberty in children and in irregular cycles, acne and male pattern body hair in adolescent girls and women (polycystic ovary syndrome). These are distressing conditions for children, who also run a higher risk of developing obesity and diabetes later in life. Male hormones are mainly produced by the gonads (ovaries in girls and testicles in boys). However, in childhood, the adrenal glands have a major role in producing male hormones.
My project will examine the mechanisms underlying the overproduction of male hormones in children. I will carry out my research with a group that has recently identified a gene defect (i.e. faulty genetic information) in a factor that is important for inactivation of male hormones. We will look for similar changes in children suffering from overproduction of such hormones. In addition, I will carry out cell culture studies to find out whether changes in the amount of this factor have a major role in the regulation of male hormones in childrens‘ adrenals. Results from this study will help to improve our understanding of mechanisms underlying male hormone overproduction and may help to develop better treatment options in the future.

Androgen excess is a key feature of the polycystic ovary syndrome (PCOS), a common endocrine disorder affecting up to 10% of females of reproductive age and rapidly emerging in childhood. Importantly, PCOS is a main risk factor for the development of the metabolic syndrome, a major driver of health care costs. In addition, girls with premature pubarche (precocious pseudo-puberty), ie the appearance of pubic hair before the age of 8 years, have been shown to be at an increased risk of developing PCOS including metabolic complications.
Recently, the investigation of a girl with premature pubarche and subsequent progression to early-onset PCOS by our group has revealed a novel monogenic cause of androgen excess, PAPSS2 deficiency. PAPS (3‘-phosphoadenosine-5‘-phosphosulfate) is the universal sulfate donor required for the sulfation of DHEA to DHEAS by DHEA sulfotransferase (SULT2A1). PAPS is generated by PAPS synthase (PAPSS) and in humans two isoforms exist: PAPSS1 is ubiquitously expressed and PAPSS2 mainly in liver and adrenal, the major sites of DHEA sulfation. Mutations in PAPSS2 in our patient lead to impaired sulfation of DHEA, which in turn directs more DHEA molecules to active androgens, thereby driving androgen excess.
In the first part of my project I will examine the role of impaired DHEA sulfation due to defects in PAPSS2 and other members of the DHEA sulfation system in children with premature adrenarche and/or early-onset PCOS. Following screening for an increased serum DHEA/DHEAS ratio I will investigate DHEA sulfation capacity in vivo, employing an oral DHEA challenge test. I will undertake sequence analysis and functionally characterise confirmed mutants in a coupled assay employing bacterially expressed PAPSS2 and SULT2A1.
Secondly, I will examine underlying molecular mechanism in more detail and address the question why ubiquitously expressed PAPSS1 cannot compensate for defect PAPSS2. Firstly, I will concomitantly over-express SULT2A1 with PAPSS1 or PAPSS2 in HEK293 cells, followed by DHEA-DHEAS conversion assays. Expression levels and subcellular localisation of the DHEA sulfation chain will be investigated by real-time PCR, Western blot and immunoflourescence to detect (co-)regulatory mechanisms. This will be complemented by siRNA knock down studies in the adrenal cell line NCIh295R that has a steroid phenotype resembling adrenal zona reticularis cells, the major site of DHEA generation.
The above programme will offer me a comprehensive, integrated laboratory and paediatric-clinical research training and potentially identify novel therapeutic targets for childhood androgen excess states.