Fig. 6 ∣. GNA11 somatic mutations were found in adrenals adjacent to double-mutant APAs.

a–c, Patient no. 7. a, Genomic DNA from six different regions (R1–R6) in the fresh-frozen adrenal sample, and associated RNA from regions 1–3 (R1–R3), were genotyped for CTNNB1 and GNA11 mutations. b, qPCR of samples in a showed 135–151-fold lower mRNA expression level of CYP11B2 and 16,102–23,987-fold lower mRNA expression level of LHCGR in R1 cDNA compared to R2 and R3, respectively. DEG highly expressed in double-mutant APAs but lowly expressed in R1 cDNA are presented in Supplementary Fig. 6a. c, Sanger sequencing of samples in a detected solitary GNA11 Q209H mutation in R1 cDNA and double mutations CTNNB1 S45F and GNA11 Q209H in R2 and R3 cDNA. Interestingly, genotyping of R1 genomic DNA (from the same sample as R1 cDNA) detected a homozygous GNA11 Q209H mutation (Supplementary Fig. 6a). d–f, Patient no. 1. d, Patient no. 1 was found to have hyperplastic ZG in adrenal adjacent to double-mutant APA; ZG hyperplasia was demarcated by lack of subcapsular CYP11B1 (visualized using a custom antibody). The hyperplastic ZG was CYP11B2 negative (visualized using a custom antibody) but LHCGR positive (visualized using the antibody NLS1436). This phenotype was consistently present in the UK/Irish discovery cohort (Supplementary Fig. 5c). e, Genomic DNA from the hyperplastic ZG of nine distinct regions of patient no. 1’s adjacent adrenal were collected systematically using segmental LCM of FFPE adrenal sections stained with cresyl violet. f, Solitary heterozygous and solitary homozygous GNA11 Q209P somatic mutations were detected in LCM ZG genomic DNA collected in e from R1 (ZG1 genomic DNA) and R6 (ZG6 genomic DNA), respectively. ZG samples from other regions were wild type for both CTNNB1 and GNA11, along with the other adrenal zones (Supplementary Fig. 6b). H&E, hematoxylin and eosin.