The PCOS GWAS Candidate Gene ZNF217 Influences Theca Cell Expression of DENND1A.V2, CYP17A1, and Androgen Production.

Polycystic ovary syndrome (PCOS), a common endocrine disorder of women, is characterized by increased ovarian androgen production and anovulatory infertility. Genome-wide association studies (GWAS) have identified more than 20 PCOS candidate loci. One GWAS candidate locus encompasses ZNF217, a zinc finger transcription factor. Immunohistochemical staining of ovarian tissue demonstrated significantly lower staining intensity for ZNF217 protein in PCOS theca interna compared to ovarian tissue from normal ovulatory women. Immunofluorescence staining of normal and PCOS theca cells demonstrated nuclear localization of ZNF217, with lower intensity in PCOS cells. Western blotting showed reduced ZNF217 protein in PCOS theca cells compared to normal theca cells, and that treatment with forskolin, which mimics the action of luteinizing hormone (LH), reduces ZNF217 expression. Lower ZNF217 expression in PCOS theca cells was confirmed by quantitative reverse transcription polymerase chain reaction. Notably, there was an inverse relationship between ZNF217 messenger RNA (mRNA) levels and theca cell androgen (dehydroepiandrosterone; DHEA) synthesis. The abundance of mRNA encoding a splice variant of DENND1A (DENND1A.V2), a PCOS candidate gene that positively regulates androgen biosynthesis, was also inversely related to ZNF217 mRNA levels. This relationship may be driven by increased miR-130b-3p, which targets DENND1A.V2 transcripts and is directly correlated with ZNF217 expression. Forced expression of ZNF217 in PCOS theca cells reduced androgen production, CYP17A1 and DENND1A.V2 mRNA, while increasing mIR-130b-3p. Conversely, knockdown of ZNF217 in normal theca cells with short hairpin RNA-expressing lentivirus particles increased DENND1A.V2 and CYP17A1 mRNA. These observations suggest that ZNF217 is part of a network of PCOS candidate genes regulating thecal cell androgen production involving DENND1A.V2 and miR-130b-3p.

Morphologic and Molecular Characterization of Adrenals and Adrenal Rest Affected by Congenital Adrenal Hyperplasia.

Introduction: Adrenocortical hyperplasia and adrenal rest tumor (ART) formation are common in congenital adrenal hyperplasia (CAH). Although driven by excessive corticotropin, much is unknown regarding the morphology and transformation of these tissues. Our study objective was to characterize CAH-affected adrenals and ART and compare with control adrenal and gonadal tissues. Patients/Methods: CAH adrenals, ART and control tissues were analyzed by histology, immunohistochemistry, and transcriptome sequencing. We investigated protein expression of the ACTH receptor (MC2R), steroidogenic (CYP11B2, CYP11B1, CYB5A) and immune (CD20, CD3, CD68) biomarkers, and delta-like 1 homolog (DLK1), a membrane bound protein broadly expressed in fetal and many endocrine cells. RNA was isolated and gene expression was analyzed by RNA sequencing (RNA-seq) followed by principle component, and unsupervised clustering analyses. Results: Based on immunohistochemistry, CAH adrenals and ART demonstrated increased zona reticularis (ZR)-like CYB5A expression, compared to CYP11B1, and CYP11B2, markers of zona fasciculata and zona glomerulosa respectively. CYP11B2 was mostly absent in CAH adrenals and absent in ART. DLK1 was present in CAH adrenal, ART, and also control adrenal and testis, but was absent in control ovary. Increased expression of adrenocortical marker MC2R, was observed in CAH adrenals compared to control adrenal. Unlike control tissues, significant nodular lymphocytic infiltration was observed in CAH adrenals and ART, with CD20 (B-cell), CD3 (T-cell) and CD68 (macrophage/monocyte) markers of inflammation. RNA-seq data revealed co-expression of adrenal MC2R, and testis-specific INSL3, HSD17B3 in testicular ART indicating the presence of both gonadal and adrenal features, and high expression of DLK1 in ART, CAH adrenals and control adrenal. Principal component analysis indicated that the ART transcriptome was more similar to CAH adrenals and least similar to control testis tissue. Conclusions: CAH-affected adrenal glands and ART have similar expression profiles and morphology, demonstrating increased CYB5A with ZR characteristics and lymphocytic infiltration, suggesting a common origin that is similarly affected by the abnormal hormonal milieu. Immune system modulators may play a role in tumor formation of CAH.

Androgen excess and diagnostic steroid biomarkers for nonclassic 21-hydroxylase deficiency without cosyntropin stimulation.

OBJECTIVES: The clinical presentation of patients with nonclassic 21-hydroxylase deficiency (N21OHD) is similar with that for other disorders of androgen excess. The diagnosis of N21OHD typically requires cosyntropin stimulation. Additionally, the management of such patients is limited by the lack of reliable biomarkers of androgen excess. Herein, we aimed to: (1.) compare the relative contribution of traditional and 11-oxyandrogens in N21OHD patients and (2.) identify steroids that accurately diagnose N21OHD with a single baseline blood draw. DESIGN: We prospectively enrolled patients who underwent a cosyntropin stimulation test for suspected N21OHD in two tertiary referral centers between January 2016 and August 2019. METHODS: Baseline sera were used to quantify 15 steroids by liquid chromatography-tandem mass spectrometry. Logistic regression modeling was implemented to select steroids that best discriminate N21OHD from controls. RESULTS: Of 86 participants (72 females), median age 26, 32 patients (25 females) had N21OHD. Age, sex distribution, and BMI were similar between patients with N21OHD and controls. Both testosterone and androstenedione were similar in patients with N21OHD and controls, while four 11-oxyandrogens were significantly higher in patients with N21OHD (ratios between medians: 1.7 to 2.2, P < 0.01 for all). 17α-Hydroxyprogesterone (6.5-fold), 16α-hydroxyprogesterone (4.1-fold), and 21-deoxycortisol (undetectable in 80% of the controls) were higher, while corticosterone was 3.6-fold lower in patients with N21OHD than in controls (P < 0.001). Together, baseline 17α-hydroxyprogesterone, 21-deoxycortisol, and corticosterone showed perfect discrimination between N21OHD and controls. CONCLUSIONS: Adrenal 11-oxyandrogens are disproportionately elevated compared to conventional androgens in N21OHD. Steroid panels can accurately diagnose N21OHD in unstimulated blood tests.

Measurement of serum tenascin-X in patients with congenital adrenal hyperplasia at risk for Ehlers-Danlos contiguous gene deletion syndrome CAH-X.

OBJECTIVE: Approximately 10% of patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency carry a mutation that disrupts CYP21A2 and the flanking TNXB gene resulting in CAH-X, a contiguous gene deletion syndrome. TNXB encodes tenascin-X (TNX), an extracellular matrix glycoprotein that plays an important role in collagen organization. TNXB impairment is associated with Ehlers-Danlos syndrome. Symptoms include joint hypermobility, hernias and cardiac defects. We measured serum TNX using an antibody targeting the amino-terminal of the TNX protein in 161 subjects, including extensively genotyped and phenotyped CAH patients, their relatives, and healthy controls. RESULTS: We evaluated the potential of serum TNX as a screening tool for CAH-X. CAH-X patients, especially haploinsufficient patients carrying the TNXA-TNXB chimeric gene CAH-X-CH-1 showed reduced TNX levels compared to controls (P < 0.05). TNX levels were similar in all subjects carrying a TNXB mutation. However, CAH patients who did not harbor a TNXB mutation also had reduced TNX compared to controls (P < 0.001). Thus, measuring serum TNX is not an effective screen for CAH-X amongst patients with CAH. TNXB genotyping is recommended for CAH patients who have symptoms of a connective tissue disorder. Epigenetic factors that influence TNX expression require further study.

The Selective RhoA Inhibitor Rhosin Promotes Stress Resiliency Through Enhancing D1-Medium Spiny Neuron Plasticity and Reducing Hyperexcitability.

BACKGROUND: Nucleus accumbens dopamine 1 receptor medium spiny neurons (D1-MSNs) play a critical role in the development of depression-like behavior in mice. Social defeat stress causes dendritic morphological changes on this MSN subtype through expression and activation of early growth response 3 (EGR3) and the Rho guanosine triphosphatase RhoA. However, it is unknown how RhoA inhibition affects electrophysiological properties underlying stress-induced susceptibility. METHODS: A novel RhoA-specific inhibitor, Rhosin, was used to inhibit RhoA activity following chronic social defeat stress. Whole-cell electrophysiological recordings of D1-MSNs were performed to assess synaptic and intrinsic consequences of Rhosin treatment on stressed mice. Additionally, recorded cells were filled and analyzed for their morphological properties. RESULTS: We found that RhoA inhibition prevents both D1-MSN hyperexcitability and reduced excitatory input to D1-MSNs caused by social defeat stress. Nucleus accumbens-specific RhoA inhibition is capable of blocking susceptibility caused by D1-MSN EGR3 expression. Lastly, we found that Rhosin enhances spine density, which correlates with D1-MSN excitability, without affecting overall dendritic branching. CONCLUSIONS: These findings demonstrate that pharmacological inhibition of RhoA during stress drives an enhancement of total spine number in a subset of nucleus accumbens neurons that prevents stress-related electrophysiological deficits and promotes stress resiliency.

Simulated visual impairment leads to cognitive slowing in older adults.

PURPOSE: To investigate the impact of different levels of simulated visual impairment on the cognitive test performance of older adults and to compare this with previous findings in younger adults. METHODS: Cognitive performance was assessed in 30 visually normal, community-dwelling older adults (mean = 70.2 ± 3.9 years). Four standard cognitive tests were used including the Digit Symbol Substitution Test, Trail Making Tests A and B, and the Stroop Color Word Test under three visual conditions: normal baseline vision and two levels of cataract simulating filters (Vistech), which were administered in a random order. Distance high-contrast visual acuity and Pelli-Robson letter contrast sensitivity were also assessed for all three visual conditions. RESULTS: Simulated cataract significantly impaired performance across all cognitive test performance measures. In addition, the impact of simulated cataract was significantly greater in this older cohort than in a younger cohort previously investigated. Individual differences in contrast sensitivity better predicted cognitive test performance than did visual acuity. CONCLUSIONS: Visual impairment can lead to slowing of cognitive performance in older adults; these effects are greater than those observed in younger participants. This has important implications for neuropsychological testing of older populations who have a high prevalence of cataract.