**2. Results**

## *2.1. Expression of CRY1, CRY2, HSD3B1, and HSD3B2 in Adrenal Tissues*

The expression levels of *CRY1*, *CRY2*, *HSD3B1*, and *HSD3B2* were determined by real-time PCR in a cohort of 46 sporadic APAs, and 20 paired adjacent adrenal tissues. Within the same sample, the median expression of *CRY1* was 2.1-fold [1.45–2.87] higher than that of *CRY2*, consistently in both APA and UAH (Figure 1A). In our cohort, the expression of both *CRY* genes was neither associated with the cellular composition of the APAs (*CRY1* expression in ZG-like APAs: 1.46 [0.45–2.59], *CRY1* expression in ZF-like APAs: 0.96 [0.49–1.58], *p*-value 0.291; *CRY2* expression in ZG-like APAs: 1.24 [0.57–2.05], *CRY2* expression in ZF-like APAs: 0.84 [0.62–1.28], *p*-value 0.170) nor with the mutational status (*CRY1* expression in wild-type APAs: 1.39 [0.58–2.7], *CRY1* expression in *KCNJ5* mutant APAs: 0.89 [0.42–1.70], *CRY1* expression in *ATP1A1-ATP2B3* mutant APAs: 0.89 [0.49–1.09], *CRY1* expression in *CACNA1D* mutant APAs: 1.4 [0.64–2.61], *p*-value = 0.417; *CRY2* expression in wild-type APAs: 1.08 [0.61–1.98], *CRY2* expression in *KCNJ5* mutant APAs: 0.98 [0.54–1.41], *CRY2* expression in *ATP1A1-ATP2B3* mutant APAs: 0.89 [0.62–1.06], *CRY2* expression in *CACNA1D* mutant APAs: 1.61 [0.80–2.87], *p*-value = 0.170). While the median expression of *CRY1* was 1.7-fold [0.75–2.26] higher in APA tissues when compared with that in the adjacent adrenal cortex (*p* = 0.016), *CRY2* showed a 20% lower expression [0.80, 0.52–1.08] in the nodule when compared with the corresponding surrounding tissue (*p* = 0.04) (Figure 1B). Representative immunohistochemistry staining of frozen tissue sections showing the expression of *CRY1* and *CRY2* in APA and adjacent adrenal cortex is illustrated in Figure 2A–F.

In both APA and UAH samples, *HSD3B2* was the main isoform, with an overall median expression 317-fold [200–573] higher than that of *HSD3B1* (*p* < 0.001) (Figure 1C). *HSD3B1* transcription was significantly more abundant (median fold change 5.2, *p* < 0.001) in APAs that were composed mainly of ZG-like cells when compared with APAs that had a ZF-like morphology (Figure 1D). A tendency towards a higher *HSD3B2* expression in APAs composed mainly of ZG-like cells was observed, but the difference did not reach statistical significance (median fold change 1.8, *p* = 0.051) (Figure 1E). In addition, the median *HSD3B1*/*HSD3B2* relative ratio was 1.9-fold higher in APA samples composed mainly of ZG-like cells (*p* = 0.003) when compared with APAs composed mainly of ZF-like cells (Figure 1F). No differences in the expression of *HSD3B1* or *HSD3B2*, according to the mutational status (*HSD3B1* expression in wild-type APAs: 1.37 [0.42–4.5], *HSD3B1* expression in *KCNJ5* mutant APAs: 0.51 [0.30–1.97], *HSD3B1* expression in *ATP1A1-ATP2B3* mutant APAs: 0.66 [0.27–1.63], *HSD3B1* expression in *CACNA1D* mutant APAs: 2.27 [1.40–3.12], *p*-value = 0.212; *HSD3B2* expression in wild-type APAs: 1.0 [0.75–2.37], *HSD3B2* expression in *KCNJ5* mutant APAs: 1.01 [0.68–2.18], *HSD3B2* expression in *ATP1A1-ATP2B3* mutant APAs: 0.69 [0.46–0.93], *HSD3B2* expression in *CACNA1D* mutant APAs: 2.82 [1.16–3.19], *p*-value = 0.147) or the final diagnosis (*HSD3B1* expression in APAs: 1.37 [0.30–3.00], *HSD3B1* expression in UAHs: 0.66 [0.42–3.61], *p*-value 0.899; *HSD3B2* expression in APAs: 1.17 [0.66–2.63], *HSD3B2* expression in UAHs: 0.99 [0.71–1.63], *p*-value 0.523), were observed. Notably, both *HSD3B1* and *HSD3B2* were significantly more expressed in the main nodule when compared with adjacent adrenal tissue (5.7- and 3.5-fold, respectively, *p* < 0.001 and *p* = 0.001) (Figure 1G). Representative immunohistochemistry staining of frozen tissue sections showing the expression of *HSD3B1* and *HSD3B2* in APAs, according to the cellular composition, is illustrated in Figure 3A–D.

**Figure 1.** *Cont.*

**Figure 1.** (**A**) Relative quantification of cryptochrome-1 (*CRY1*) mRNA over cryptochrome-2 (*CRY2*) messenger RNA (mRNA) in the total cohort, in aldosterone-producing adenoma (APA) samples (*n* = 35) and in unilateral adrenal hyperplasia (UAH) samples (*n* = 11). (**B**) Relative quantification of *CRY1* and *CRY2* mRNA in APA samples over that in the corresponding adjacent adrenal cortex (*n* = 20). (**C**) Relative quantification of type II 3β-hydroxyl-steroid dehydrogenase (*HSD3B2*) mRNA over type I 3β-hydroxyl-steroid dehydrogenase (*HSD3B1*) mRNA in the total cohort, in APA samples (*n* = 35) and in UAH samples (*n* = 11). (**D**) *HSD3B1* mRNA expression according to the cellular composition in the total cohort of adrenal samples. (**E**) *HSD3B2* mRNA expression according to the cellular composition in the total cohort of adrenal samples. (**F**) Relative quantification of *HSD3B1* mRNA over *HSD3B2* mRNA according to the cellular composition. (**G**) Relative quantification of *HSD3B1* and *HSD3B2* mRNA in APA samples over that in the corresponding adjacent adrenal cortex. For each box plot, the horizontal line represents the median, and the box and bar indicate the 25th to 75th and 5th to 95th percentiles, respectively.
