THE MUTATIONAL LANDSCAPE OF ADRENAL GLANDS WITH ALDOSTERONE-PRODUCING LESIONS AT SINGLE-CELL RESOLUTION

Objective: Aldosterone-producing micronodules (APMs), clusters of cells in the subcapsular regions of the adrenal cortex that autonomously express aldosterone synthase (encoded by the gene CYP11B2), are potential precursors to aldosterone-producing adenomas (APAs), which leads to excess aldosterone production and secondary hypertension. Increased mutation burden has been well-associated with aging and disease development. APMs also seem to increase with age and harbor aldosterone-driving mutations. To further investigate the driver for APMs development, we assessed the somatic mutational burden of normal adrenal tissue adjacent (NAA) to APAs and non-APA adenomas using single-cell RNA sequencing data. Design and method: Single-cell RNA sequencing of adrenal tissues from eight patients who underwent adrenalectomy at St Bartholomew’s Hospital, London, United Kingdom, and Changi General Hospital, Singapore was performed using 10x Genomics platform. Single nucleotide variants to assess mutational burden were identified using the SComatic algorithm. This method was also repeated on publicly available 10x single-cell datasets from Japan. Results: In this study, adrenal samples of varied ethnic origin and disease phenotype were used. Notably, a higher mutation frequency was observed in Singapore patients compared to London patients. The predominant cell types identified in the adrenals were steroidogenic cells, immune cells, endothelial cells, and smooth muscle cells. Interestingly, the mutation burden was most pronounced in adrenocortical zona glomerulosa (ZG) cells from Asian patients. Moreover, missense mutations identified in NAA to APAs of Asian patients were predominantly detected in ZG cells. These include mutations in CYP11B2, the gene for the key enzyme for aldosterone production, and members of the ubiquitin-specific protease (USP) gene family. Interestingly, mutations in USPs have been reported to frequently occur in Cushing’s disease, where there is an excess of another adrenal steroid, cortisol. Conclusions: In depth insights into the somatic mutation burden of different cell subtypes within heterogeneous adrenal tissues may elucidate the driver for development of autonomous aldosterone-producing cells. We postulate that the cells with a high mutational burden accumulate aldosterone-driving somatic mutations, contributing to the development of APMs. Interrogation of larger datasets are needed to verify this and to elucidate further if APAs genotype may impact these findings.