Increased expression of ACTH (MC2R) and androgen (AR) receptors in giant bilateral myelolipomas from patients with congenital adrenal hyperplasia

After surgical resection, tumor fragments were immediately frozen in liquid nitrogen and stored at -80°C until total RNA extraction using Trizol reagent (Invitrogen, Carlsbad, CA). cDNA was generated using a High Capacity kit (Applied Biosystems, Foster City, CA, USA). Quantitative real-time PCR (qRT-PCR) was performed with an ABI Prism 7700 sequence detector using TaqMan gene expression assays (Applied Biosystems, Foster City, CA). The assay identifications were the following: MC2R (Hs00265039_s1), AR (Hs00907244_m1), SF1 (Hs00610436_m1) and LEP (Hs01084494_m1). Beta-actin (ACTB) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were used as endogenous genes. A commercial pool of adipose tissue and adrenal tissue was used for comparisons (CLONTECH, BioChain, and Ambion). Relative quantification was performed using the 2^-ΔΔCT method [12]. Overexpression was defined as a two-fold change in comparison to normal adipose tissue.

Genomic DNA was obtained from tumors using standard procedures. The X-chromosome inactivation pattern and CAG repeat numbers were determined as previously described [13]. Briefly, PCR amplification of the CAG repeat region of HpaII- digested and undigested samples was carried out using primers flanking the region of interest: 5’-GCTGTGAAGGTTGCTGTTCCTC-3’ and 5'-HEX-GTGCGCGAAGTGATCCAGAA-3’. All samples were separately amplified in 50 μL reactions containing 1x PCR reaction buffer with 1.5 mM of MgCl₂, 200 μM of deoxynucleotides, 15 pmol of each primer and 1 U of Taq DNA Polymerase (Amersham-Pharmacia, Uppsala, Sweden). Amplifications were performed under the following conditions: initial denaturation at 97°C for 5 min; amplification for 35 cycles with denaturation at 97°C for 1 min, annealing at 54°C for 45 s and extension at 72°C for 45 s; and one final extension at 72°C for 30 min. Two and 4 μL of PCR products from undigested and digested samples, respectively, were submitted to capillary electrophoresis on ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) and analyzed by GeneScan software to determine the sizes of the amplified fragments, which were established through comparisons with a size marker and a sample with a known CAG repeat number in the same run. These sizes were correlated with CAG repeat numbers, as previously shown in our lab [14]. Digested and undigested samples were also assayed in the same run, and the peak height of each allele was used to determine the X-chromosome inactivation pattern.

Patient 1 was a 35-yr-old woman who presented with the simple virilizing form of CAH [p.E351V and exon 6 cluster (p.I236N, p.V237E, and p.M239K) mutations in a compound heterozygote state in the CYP21A2 gene]. CT scan revealed giant bilateral adrenal myelolipomas (left, 14 × 14 × 10 cm; right 8.9 × 8.3 × 8.0 cm) with fat component density. She did not present to clinical follow-up and had not received any medications in the last 15 yr. Hormonal evaluation revealed extremely increased levels: basal 17OH-progesterone (17-OHP) 192 ng/mL, ACTH 1,172 pg/mL, total testosterone 949 ng/dL and androstenedione 17 ng/mL (Figures 1A and 1B).

Patient 2 was a 52-yr-old woman who presented with the simple virilizing form of CAH (IVS2-13A/C > G/ p.I172N). CT scan revealed giant bilateral adrenal myelolipoma (left, 16 × 13 × 9.0 cm; right, 5.3 × 4.3 × 6.9 cm) with fat component density (Figures 1C and 1D). This patient had never been previously treated, and hormonal evaluation also revealed increased serum levels: 17-OHP 120 ng/mL, total testosterone 720 ng/dL and androstenedione 39 ng/mL. ACTH measurement was not available. Both CAH patients were severely virilized during adolescence and changed to male social sex. They sought medical assistance due to abdominal pain.

Briefly, the histopathological analysis of all tumors showed lobes of mature adipose tissue mixed with abundant hematopoietic tissue consisting of all three hematopoietic elements with mature and precursor cells. There were islets of cells from the zones of the adrenal cortex in the periphery of tumors as well as between hematopoietic and lipoid cells.

The MC2R gene was overexpressed in the myelolipomas of 3 out of 4 patients (Table 1). MC2R expression was correlated with SF1 mRNA levels in the same tumors. Only the myelolipoma diagnosed in patient 1 did not show MC2R overexpression. As expected, MC2R mRNA levels were higher in the adrenal pool.

The AR gene was overexpressed in myelolipomas from patients 2 and 4. Low AR expression levels were found in the other cases (patients 1 and 3). AR expression was correlated with LEP expression in all tumors.

X-chromosome inactivation pattern analysis revealed a polyclonal origin in all tumors. In addition, all tumors were informative, and nCAG repeat numbers varied from 20 to 30, except in the tumor from patient 1, which had a short allele (15 repeats) (Table 1).