Primary adrenal insufficiency (PAI) is a potentially life-threatening condition that can present with non-specific features and can be difficult to diagnose. Here, we undertook next-generation sequencing in a cohort of children and young adults with PAI of unknown etiology from around the world and identified a heterozygous missense variant (rs6161, c.940G>A, p.Glu314Lys) in CYP11A1 in 19 individuals from 13 different families (allele frequency within undiagnosed PAI in our cohort 0.102 vs 0.0026 in gnomAD, pA, Thr330=; c.1173C>T, Ser391=). Although p.Glu314Lys is predicted to be benign and showed no loss-of-function in an E. coli assay system, in silico and in vitro studies revealed that the rs6161/c.940G>A variant, plus the c.990G>A and c.1173C>T changes, affect splicing and that p.Glu314Lys produces a non-functional protein in mammalian cells. Taken together, these findings show that compound heterozygosity involving a relatively common and predicted “benign” variant in CYP11A1 is a significant contributor to PAI of unknown etiology, especially in European populations. These observations have implications for personalized management and demonstrate how variants that might be overlooked in standard analyses can be pathogenic when in combination with other very rare disruptive changes.

Adrenal insufficiency requires lifelong corticoid replacement therapies. However, current therapies are not able to replace the physiological circadian pattern of the adrenal cortex and are associated with many metabolic, vascular, neuroendocrine, and mental perturbations. Therefore, regenerative and more curative strategies would be desirable. In the current perspective, we describe emerging new regenerative therapies for the treatment of adrenal insufficiency. In particular, we discuss gene therapy and cell replacement strategies. Furthermore, we discuss how adrenal cells might be used as a source for regenerative therapies of nonadrenal neurodegenerative diseases such as Parkinson disease.

Background: Abnormal pubertal timing affects over 4% of adolescents and is associated with adverse health and psychosocial outcomes. Previous studies estimate that 60–80% of variation in the timing of pubertal onset is genetically determined. However, despite this strong heritability, little is known about the genetic control of human puberty. Self-limited delayed puberty (DP) segregates in an autosomal dominant pattern, but in the majority of patients the neuroendocrine pathophysiology and its genetic regulation remain unclear. Methods: We performed whole exome sequencing in 52 members of seven families from our patient cohort with DP, with follow-up targeted re-sequencing of candidate genes in a further 42 families. The functional consequences of the identified mutations in one candidate gene were interrogated via expression of WT and mutant proteins in mammalian cells. For this gene we defined tissue expression in human and mouse embryos by in situ hybridization and immunohistochemistry. The effects of gene knockdown were investigated via in vitro neuronal migration assays, and in vivo using a transgenic zebrafish model with fluorescently labeled GnRH neurons. Results: We identified four rare mutations in IGSF10 in ten unrelated families, which are tightly associated with the DP trait within our cohort (P=3.47×10⁻⁴). The identified mutations are in evolutionarily conserved positions, and two mutations result in intracellular retention with failure in secretion of the N-terminal fragment of the protein. IGSF10 mRNA is strongly expressed in the nasal mesenchyme in mouse and human embryos, during the time-period when GnRH neurons migrate from their nasal origin towards the hypothalamus. IGSF10 knockdown caused reduced migration of immature GnRH neurons in the in vitro analysis, and perturbed migration and extension of GnRH neurons in the zebrafish model. Conclusions: We present our novel finding that mutations in IGSF10 cause delayed puberty in humans, through misregulation of GnRH neuronal migration during embryonic development.

Background: The human adrenal cortex is composed of different zones, namely the zona glomerulosa (ZG), zona fasciculata (ZF), zona reticularis, and the recently proposed undifferentiated zone (UZ). The adrenal cortex synthesises and secretes steroids, mainly aldosterone and cortisol, both responsible for essential physiological and metabolic functions. Adrenal cortex disorders can be life threatening and current treatment involves life-long steroid replacement, which is not ideal. Aim: We aim at identifying markers to distinguish (and eventually isolate) cells from the different zones, with a specific focus on the subcapsular region. Methods: Following ethical approval, adrenal glands were collected from patients undergoing adrenalectomy, snap–frozen in liquid nitrogen and cut using a cryostat. We tested a wide array of antibodies to antigens known to have a zonal expression in murine studies. Non-radioactive in situ hybridization was also employed. Results: We demonstrated that the human ZU is a more complex environment than we had anticipated. Delta-like homologue-1 (DLK1) seems to be a good marker of the ZU; however we found clusters of non-steroidogenic cells in the subcapsular region that are DLK1 negative and we are currently searching for specific markers. Conclusion: The detailed characterisation of the subcapsular region in the human adrenal will provide invaluable information of steroidogenic differentiation processes and tissue homeostasis. It will also aid in the isolation of cells from the different zones using laser capture microdissection followed by important downstream applications, such as a transcriptome comparative analysis.

Background: CYP11A1 encodes the P450 side chain cleavage enzyme (P450scc) responsible for initiating steroidogenesis and classically gives rise to disordered sex development plus adrenal and gonadal insufficiency. The rs6161 variant in exon 5 of CYP11A1 (c.940G>A; p.E314K) has previously been considered as ‘benign’. When next generation sequencing was performed in patients with primary adrenal insufficiency of unknown etiology the rs6161 variant was found as a heterozygous change in 15 subjects (minor allele frequency 0.0625 (our cohort) vs 0.0024 (ExAC database)). In 13 patients the c.940G>A variant occurred in trans with a second clearly disruptive heterozygous change, in the remaining two patients the only other novel variant was a synonymous substitution (c.990G>A (p.T330=), c.1173C>T (p.S391=)). Given the enrichment of the c.940A allele and rarity of the two synonymous variants c.990A and c.1173T (MAF 0 and 3×10⁶ respectively), we suspected they were pathogenic and therefore investigated their effects on mRNA processing and/or protein function. Results: An in vitro splicing assay demonstrated aberrant splicing for all three variants, causing exon 5 skipping in most RNA transcripts for variants c.940A and c.990A and complete exon 7 skipping for c.1173T. In each instance when the exon is skipped a frameshift and premature translation-termination codon will occur. We corroborated the findings for variants c.940A and c.990A in vivo in patient samples. Mutant p.314K P450scc enzyme activity did not differ from WT in a validated functional assay in E. coli. However, when expressed in a eukaryotic cell line the mutant protein is truncated to 30-35 kDa, and the half-life is much shorter than for WT. Conclusion: This comprehensive analysis illustrates how multiple mechanisms might contribute to loss-of-function and highlights the fact that in silico prediction tools are inadequate. Therefore functional analyses need to be undertaken on all rare variants to determine pathogenicity and correctly assign clinical significance.

Cellular reprogramming describes the process where a fully differentiated, specialized cell type is induced to transform into a different cell. Cell reprogramming techniques can become powerful tools for modelling diseases, drug testing and for personalized cellular therapy. The adrenal cortex is the primary site of steroid synthesis. Adrenal insufficiency, which can be life threatening, is caused by a number of adrenal disorders, and lifelong management of these patients with exogenous steroids can be challenging. Our long-term goal is to develop novel personalized and curative treatments that use stem cells and cellular reprogramming to treat the many progressive and debilitating conditions affecting the adrenal cortex. Steroidogenic Factor-1 (SF1) is a transcription factor essential for both adrenal and gonadal development. SF1 positively regulates steroidogenic genes transcription and can be considered a true effector of cell fate as it starts a genetic program driving embryonic mesenchymal cells towards a steroidogenic phenotype/lineage. We have discovered that cells derived from urine (urine-derived stem cells, USCs) can be reprogrammed to a steroidogenic phenotype with a highly reproducible phenotype as assessed by changes in cell morphology, gene expression, activation of adrenal-specific signalling pathways and hormonal output. Urine is the perfect cell source reservoir as its harvest is the least invasive. In vivo transplantation experiments in nude mice using alginate-embedded reprogrammed human urine-derived steroidogenic cells are currently being performed to test the functionality of these cells in a more physiological environment. Moreover, USCs derived from patients with familial glucocorticoid deficiency (FGD) and congenital adrenal hyperplasia (CAH) are being isolated, amplified and banked and we are aiming to use the new genome editing tool CRISPR-Cas9 to repair the monogenic mutations occurring in these conditions. These will allow us to in vitro assess the hormonal profiles of affected versus CRISPR-Cas9 gene corrected cells, serving as a proof-of-concept of our technology.