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However, the single-cell mechanisms underlying this heterogeneity remain to be fully elucidated.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Design<\/jats:title>\n                    <jats:p>Multi-omics analysis.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>We conducted multi-omics analysis of adrenal tissue obtained from a 33-year-old female with PPNAD, harboring multiple pigmented nodules and a cortisol-producing adenoma (CPA).<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>Genomic analysis revealed that each nodule represents an independent clonal lesion initiated by a germline PRKAR1A mutation on one allele, followed by distinct somatic alterations on the other allele through deletion or mutation. This resulted in biallelic inactivation, thereby constitutively activating the PKA pathway. Single-cell RNA sequencing and spatial transcriptomic analysis revealed that the examined nodules exhibit features of the zona reticularis, including upregulation of genes involved in androgen synthesis, cellular senescence, and immune activation, consistent with tumor-suppressive features. A significant activation of the Wnt\/\u03b2-catenin signaling pathway associated with tumor proliferation was observed in CPA. Pseudotime trajectory analysis revealed a transcriptional trajectory consistent with a potential transition from nodule-derived cells to adenoma cells.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>We postulate that PPNAD nodules arise as multiple independent clonal lesions driven by biallelic PRKAR1A inactivation and characterized by tumor-suppressive features, and that activation of the Wnt\/\u03b2-catenin pathway is associated with progression from nodules to CPA. This study provides evidence that independent clonal evolution underlies the heterogeneity of multinodular lesions in PPNAD.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1093\/ejendo\/lvag113","type":"journal-article","created":{"date-parts":[[2026,6,26]],"date-time":"2026-06-26T20:13:52Z","timestamp":1782504832000},"page":"56-67","source":"Crossref","is-referenced-by-count":0,"title":["Independent clonal evolution in primary pigmented nodular adrenocortical disease: a single-patient multi-omics study"],"prefix":"10.1093","volume":"195","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-2870-2776","authenticated-orcid":false,"given":"Tazuru","family":"Fukumoto","sequence":"first","affiliation":[{"name":"Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University , Fukuoka 812-8582 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