MOST · Modelling Steroidogenesis

Modelling Steroidogenesis

Congenital Adrenal Hyperplasia (CAH) ranks amongst the most common inherited metabolic disorders. CAH patients commonly manifest with adrenal insufficiency and disorders of sex development (DSD) in both sexes. Steroid 21-hydroxylase deficiency (21OHD) accounts for approximately 95% of CAH cases which results from mutations in CYP21A2 gene. Steroid 21-hydroxylase facilitates the conversion of 17-hydroxyprogesterone (17OHP) to 11-deoxycortisol (S), and progesterone to deoxycorticosterone, respective precursors for cortisol and aldosterone. Approximately 75% of cases of 21OHD are unable to synthesize sufficient aldosterone and suffer from clinically apparent renal salt loss. Impaired cortisol biosynthesis leads to reduced negative feedback towards the hypothalamus and pituitary and results in increased corticotropin releasing hormone (CRH) and adrenocorticotrophic hormone (ACTH) secretion. Elevated CRH and ACTH concentrations stimulate adrenal hyperplasia and a rise in adrenal androgen production. The clinical consequences of CAH therefore result from cortisol and aldosterone insufficiency and from sex steroid excess. Glucocorticoid replacement therapy with or without mineralocorticoid supplementation usually resolves clinical symptoms. However, long-term outcome studies have shown that comorbidities are common in CAH patients and that there is a need for personalised treatment to overcome such problems. To develop novel innovative therapeutic options for CAH patients, it will be of paramount importance to gain a deeper understanding of the underlying molecular pathogenesis as well as the disease specific complications of 21OHD. Such studies will not be able in humans. Therefore the development of novel model systems to study human disease is highly promoted. Zebrafish are a comprehensive complementary in vivo model organism for studying adrenal function as its organogenesis resembles mammalian development function. The project of Dr Guran aimed to study: (1) the in vitro and in vivo function of the zebrafish 21-hydroxylase; (2) the whole organism response to complete and partial 21-hydroxylase deficiency during development and adult life. The first aim of this project involved: (i) cDNA amplification and genotyping of zCyp21a2 from interrenal gland (the adrenal counterpart of the mammalian adrenal), ovary, testis and brain tissues of the zebrafish; (ii) molecular cloning of zCyp21a2; (iii) spatio-temporal expression studies; (iv) whole-mount in situ hybridization using zCyp21a2 riboprobes on zebrafish embryos during developmental stages (iii) transient morpholino knockdown studies in zebrafish embryos, (iv) morphological characterisation of wild-type and morphant zebrafish embryos using live imaging techniques; (v) rescue trials with zCyp21a2 mRNA overexpression and dexamethasone treatments of embryos; (vi) extraction of steroids from zebrafish embryos for steroid profiling by LC-MS/MS; (vii) and functional in vitro studies employing mammalian cell lines. In this first part of her fellowship Dr Guran has generated novel data with important impacts on potential regulatory mechanisms of steroidogenesis in early development summarized as follows: o Expression of zCyp21a2 starts during late segmentation period (28 hpf), and become prominent from 32 hpf in zebrafish shown by RT-PCR (Fig1A) and in situ hybridisation (Fig1B) assays. This expression pattern is similar to that of the genes encoding key steroidogenic enzymes (zCyp11a2, zHsd3b2) which complies with the start of endogenous cortisol production in zebrafish embryos. In adult zebrafish tissues, similar to that of humans, expression of zCyp21a2 is restricted to steroidogenic tissues, namely interrenal gland, gonads and brain (Fig1C). o Unlike many steroidogenic genes in zebrafish genome, zCyp11a2 has no duplicated transcript suggesting unique physiological role in steroidogenesis. However, Dr Guran could be able to define an alternative transcript of zCyp21a2 lacking universally conserved exon 9 (zCyp21a2-e9). This exon is located in the central I helix of crystal structure of 21-hydroxylase protein and highly conserved among species and in the majority of P450 enzyme superfamily. It is involved in stabilizing the dioxygen complex, assist in proton transfer and substrate binding. Based on, Dr Guran`s data this default alternative transcript zCyp21a2-e9 is maternally inherited and is expressed all over early development (Fig1D). o Adult fish steroidogenic tissues and gonads show sexually dimorphic expression of 2 transcripts of zCyp21a2 (Fig1E). o Overexpression assays employing mammalian cell lines have demonstrated that whole length zCyp21a2 is involved in 17OHP-to-S conversion, as in humans, whereas zCyp21a2-e9 variant is non-functional (Fig1H). o The role of Cyp21a2 during development in vivo has been evaluated by generating transient knockdowns with specific anti-sense morpholinos. Two different Fdx1b morpholinos were designed: an ATG morpholino (Cyp21a2-ATGMO), and a splice morpholino (Cyp21a2-SplMO; targeting the splice acceptor site of intron four) and are injected into the yolk of 1-cell stage embryos. Stereomicroscopic observations over 120 hpf revealed distinctive phenotypical characteristics of morphants including craniofacial abnormalities, pericardial oedema, curved body and tail and abnormal pigmentation pattern (Fig1F) compared to control embryos (Fig1G). However, liquid chromatography/tandem mass spectrometry (LC-MS/MS) based steroid measurements from morphant embryos were unable to demonstrate impaired steroidogenesis. Furthermore, phenotypical changes of morphant embryos could not been rescued by injecting open-reading frames (ORFs) of wild-type zCyp21a2 and 11-deoxycortisol supplementation at titrated concentrations. Hence, forementoined changes were considered as toxic or off-target effects which subsequently led us to generate stably expressing mutant zCyp21a2 strains by TALEN genome editing approach. o Along with above-mentioned purposes, Dr Guran has designed pairs of TALENs targeting exon 2 to achieve secondary aim of the research proposal. Following the injection at 1-cell stage, fish embryos were screened for targeted indels by sequencing, high resolution melting (HRM) and enzymatic digestion studies, which were found positive in F0 and F1 strains. Encouragingly, mutant fish embryos showed hyper-pigmented phenotype, characteristic for primary adrenal insufficiency in humans. Studies for establishing sexually mature F3 generations of zCyp21a2 null alleles are still ongoing to evaluate whole organism impact of 21OHD. Thus, the work conducted during Dr Guran`s fellowship has brought significant novel insights into the regulation of steroidogenesis in zebrafish. Overall, the exciting data from this project provides further evidence for zebrafish as a cutting-edge model in translational research of adrenal disease. Furthermore, using zebrafish as a model organism will allow to study in vivo the pathophysiological response to inborn errors of steroidogenesis during early embryologic development that may determine long-term co-morbidities observed in patients with imbalanced steroidogenesis. The data produced by Dr Guran with this work has already been submitted as 2 abstracts (1- Zaucker A, Guran T, Griffin A, Krone N. Studying the consequences of disrupted steroid hormone biosynthesis on development and function of the brain. Zebrafish Symposium, 7 Nov 2014, Warwick Medical School, UK; 2- Zaucker A, Guran T, Thakur N, Taylor A, Griffin A, Krone N. A novel animal model to explore the whole-organism response to 21-hydroxylase deficiency. Annual Meeting of Society for Endocrinology BES 2015, Edinburgh, UK). Whilst continuing with the experimental programme, Dr Guran could be able to maximize the opportunities for advanced training provided by the fellowship. She worked on the functions of 5-alpha reductases in steroid metabolism under the supervision of Prof Wiebke Arlt, a leading researcher in the steroid field and specialist for adrenal and gonadal disorders. She performed functional in vitro studies of novel as well as previously described mutations in SRD5A2 gene leading to 5α-reductase 2 deficiency using mammalian stable overexpression systems, western blotting and the state-of-the-art liquid chromatography/tandem mass spectrometry (LC-MS/MS) (manuscript under preparation). She has also demonstrated, for the first time, that 5α-Reductase type 3 deficiency has no impact on steroid metabolism in humans by the state-of-the-art gas chromatography-mass spectrometry (GC-MS) (manuscript submitted to Journal of Clinical Endocrinology and Metabolism). Dr Guran learned technical details to process different matrices such as zebrafish, cell culture media, nappy and fluid urine to extract steroids. Additionally, in collaboration with Prof. Cedric Shackleton who is a pioneer in the era of GC-MS based steroid assays, she has revisited GC-MS based measurements from more than 1000 healthy controls (data obtained from Prof Wiebke Arlt`s group archive) and patients with various steroidogenic disorders (data obtained from “UK Gonadal and Adrenal Inherited disorders Network (UKGAIN)” a FP-7 project where Prof Arlt`s group is a collaborator) to improve previously established diagnostic ratios of urinary steroid metabolites for the diagnosis of the different CAH forms. On these grounds, she has acquired a broad range of analytical skills required for her development as a translational researcher on adrenal gland. This extensive data set will serve to establish unsupervised clustered model to discriminate various congenital steroidogenic disorders in collaboration with Dr Michael Biehl, an internationally leading expert in the fields of scientific computing and machine learning. Application of this method will be of huge benefit for clinical practice to outcome prediction and early diagnosis of congenital adrenal disorders. Dr Guran had the opportunity to study molecular genetic analyses of considerable numbers of patients referred to Centre of Endocrinology, Diabetes and Metabolism (CEDAM) as an institution of excellence in University of Birmingham (an example of a publication from this work: Turkkahraman D, Guran T, Ivison H, Griffin A, Vijzelaar R, Krone N. Identification of a novel large CYP17A1 deletion by MLPA analysis in a family with classic 17α-hydroxylase deficiency. Sex Dev. 2015;9:91-97). She has acquired further skills in scientific writing which represents an important aspect of the scientific training (a book chapter: Baranowski E, Guran T, Krone N. The hypothalamo-pituitary-adrenal axis and its regulation. In Neuroendocrine Disorders in Children. (in press)) Finally, Dr Guran`s work brought significant contributions into the pathophysiology of CAH and inborn error of steroidogenesis in multiple aspects besides her achievements in translational and interdisciplinary research and transferable core facilities.

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