United States

The adrenal cortex and gonads are the two major endocrine hubs that participate in the hormonal regulation of vital aspects of mammalian homeostasis. Accordingly, their developmental aberrancies drive various congenital and adult-onset diseases. Lineage tracing studies in mice reveal that the adrenal cortex and gonads originate from a common progenitor, the adrenogonadal primordium. However, the specification pathways of the adrenal cortex and gonads and their lineage relationship remain poorly understood in humans. Here, we conduct the high resolution spatial and temporal lineage trajectory mapping of early human adrenal and gonadal lineages using single cell transcriptomics and histologic profiling on human embryos at 3-8 week post fertilization. We find that in humans, the adrenocortical lineage originates in a temporally and spatially distinct fashion from the gonadal lineage, arising earlier and more anteriorly within the coelomic epithelium. The adrenal primordium arises from adrenogenic coelomic epithelium via an epithelial-to-mesenchymal-like transition, which then progresses into the steroidogenic fetal zone via both direct and indirect routes. Notably, we find that adrenocortical and gonadal lineages exhibit distinct HOX codes, suggesting distinct anterior-posterior regionalization. Together, our assessment of the early divergence of these lineages challenges the paradigm established in rodents and present evidence that the specification programs directing the formation of adrenal glands and gonads differ between rodents and humans. The molecular details of early lineage diversification of human adrenals and gonads will serve as both a framework for understanding the molecular pathology of human disorders.

ENDO 2022

The developmental origin and the specification of the adrenal cortex in humans and cynomolgus monkeys

**Program**
ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

This year’s education program features:
more than 100 in-person and virtual sessions with another 70+ in-person only sessions;
six plenary sessions including plenaries on Envisioning Future Therapies for Type 1 Diabetes and Addressing Challenges in Fertility and Reproduction;
over 20 Meet the Professor sessions presented both in-person and live-streamed virtually and another 20+ presented live in Atlanta;
four Basic Science Pathways, including Diabetes and Metabolism; Neuroendocrinology; Nuclear Receptors and Signaling; and Reproductive Endocrinology; and
more than 30 oral sessions and a robust virtual poster hall for accepted scientific abstracts.

**What Programming Is Included in My Virtual Meeting Registration?**
As you build your schedule during the conference, you can select the sessions that are tagged with 'Livestreamed', so you can view virtually. These sessions are being livestreamed, so they are available in real time to our virtual audience. In addition, our in-person and livestreamed sessions will be recorded, so you can access them on demand after 72 hours. We will have over 200 hours available on demand for Standard and Premium pass registrants.

Product theaters and the Communications and Career Center will also include hybrid content delivery.

**Plenaries**
Spanning the spectrum of science, clinical care, and social implications, an outstanding slate of world-renowned speakers will showcase the most cutting-edge advances on research and medicine.

**Symposia**
Our ENDO 2022 lineup of sessions offers a unique opportunity to dive into topics from nearly every major therapeutic area of endocrinology and hormone science. Listen, learn, and be inspired by world-class faculty who are at the forefront of scientific discovery and high-quality health care.

**Basic Science Pathways**

These special “meeting-within-a-meeting” tracks integrate focused discussions, poster sessions, and networking opportunities to provide you with the latest research in:
Diabetes and Metabolism
Neuroendocrinology
Nuclear Receptors and Signaling
Reproductive Endocrinology

**Meet the Professor & Meet the Scientist**
Discover insights from experts on treating a variety of endocrine conditions and emerging science in the field of endocrinology, and learn the latest in patient care and scientific research.

ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

technical paper

Proper development and zonation of the adrenal cortex is intimately connected with its lifelong capacity to produce steroid hormones. Both adrenocortical development and homeostasis are mediated in part by paracrine WNT/β-catenin signaling, an essential pathway for zonal maintenance and aldosterone production. Patients harboring homozygous or compound heterozygous loss-of-function (LOF) mutations in the WNT ligand WNT2B present with congenital diarrhea and require parenteral nutrition. Despite evidence for euvolemia, WNT2B-null patients exhibit markedly elevated plasma renin levels with no corresponding increase in aldosterone, suggesting a primary adrenal defect in aldosterone synthesis. To test this hypothesis, we generated both global and conditional Wnt2b knockout mouse models. In the mouse adrenal, Wnt2b is expressed exclusively in the capsule, the outer compartment of mesenchymal cells that overlay and signal to the subcapsular zona glomerulosa (zG), the adrenocortical zone that harbors aldosterone-producing cells and critical long-term progenitor cells. Global loss of Wnt2b from early mouse development results in a near-complete absence of the histological zG. Moreover, Wnt2b-null mice exhibit significantly increased plasma renin but normal aldosterone levels compared to wild-type controls. These data suggest that WNT2B-null patients indeed have an adrenocortical phenotype caused by apparent zG hypofunction. To further define the mechanism(s) by which Wnt2b LOF leads to zG hypofunction, we temporally deleted Wnt2b in the adrenal capsule of adult mice. Wnt2b conditional knockout (cKO) adrenals exhibit a zG-restricted decrease in Wnt/β-catenin signaling as determined by decreased β-catenin activity and target gene expression. Moreover, Wnt2b cKO mice demonstrated a disorganized zG and disrupted adrenocortical zonation. To determine the contribution of WNT2B to aldosterone production, we administered a sodium-deficient diet to control and Wnt2b cKO mice to activate the renin-angiotensin-aldosterone system. Indeed, while control mice exhibited the expected increase in both plasma renin and aldosterone, Wnt2b cKO mice showed marked elevation of renin despite normal aldosterone compared to controls, consistent with hyperreninemic hypoaldosteronism. Together, our work supports a WNT2B-dependent mechanism for adrenal zG development and maintenance in mice and humans and provides relevant models to study the consequences of WNT2B deficiency within the zG.

WNT2B Deficiency Results in Hyperreninemic Hypoaldosteronism in Humans and Mice

Introduction: Primary Bilateral Macronodular Adrenal Hyperplasia (PBMAH) is a heterogeneous disease characterized by bilateral adrenal macronodules responsible for adrenal Cushing. To date, two genetic causes of PBMAH are known: germline inactivating variants of the tumor suppressor genes ARMC5 identified in 2013 (Assié, N Eng J Med 2013), responsible for 20 to 25% of index cases, and KDM1A, identified recently (Vaczlavik, GIM 2021; Chasseloup, Lancet D&E 2021), responsible for the rare presentation associated with food-dependent Cushing's syndrome (FDCS) due to aberrant expression of the GIP receptor (GIPR) in adrenocortical cells. Multiple other illegitimate receptors are known to be responsible for abnormal cortisol response to various physiological stimuli in PBMAH. A recent multiomic analysis, identified three distinct molecular PBMAH groups: G1 with ARMC5-mutated tumors, G2 with KDM1A-mutated tumors from FDCS patients, and G3 with no identified genetic cause at present. We aimed to identify specific expression profiles of illegitimate receptors in the three groups.
Methods: Based on the transcriptome data obtained by RNA sequencing (Illumina) of the tumors from 31 patients (G1/ARMC5, 16 patients; G2/KDM1A, 6 patients; G3, 9 patients), expression of the following genes, encoding potential illegitimate receptors, were compared: ADRA1A, ADRA1B, ADRA1D, ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, ADRB3, AVPR1A, AVPR1B, AVPR2, GCGR, GIPR, HTR4, HTR7, LHCGR. Calculations were performed using R statistical software. The Bioconductor limma package was used to analyze mRNA differential expression.
Results: G1/ARMC5 tumors showed a relative overexpression of the vasopressin receptors AVPR1A and AVPR1B compared to the two other groups (fold-change [FC] =7.39, p<0.001 and 3.98, p<0.001, respectively) but a lower expression of AVPR2 (FC=0.43, p=0.015). G2/KDM1A tumors showed a dramatic overexpression of GIPR compared to the two other groups (FC=105.02, p<0.001) but also of the adrenergic receptors ADRA1D and ADRA2A (FC=2.93, p=0.027 and 9.99, p<0.001, respectively) and of the LH/hCG receptor (LHCGR) (FC=12.20, p<0.001). G3 tumors showed a slight overexpression of the adrenergic receptor ADRA1B (FC=3.49, p=0.001) and in few tumors ADRA1D, AVPR2 and LHCGR were highly expressed suggesting molecular heterogeneity in G3.
Conclusion: This study reveals specific expression profiles of illegitimate receptors related to the three molecular groups. ARMC5 tumors are associated with the overexpression of two vasopressin receptors, while, besides GIPR, KDM1A inactivation seems to drive the overexpression of the LH/hCG receptor, as previously suggested in patients with FDCS (Bertherat, JCE&M 2005), potentially responsible for Cushing's syndrome associated with pregnancy and menopause. These molecular patterns need to be corroborated by clinical data with a systematic testing of the aberrant cortisol responses. Additionally, further studies would be needed to investigate the clinical relevance and significance of moderate fold-changes in gene expression (e.g. <4).

Genetic Alterations of ARMC5 and KDM1A Are Associated With Different Expression Profiles of Illegitimate Receptors in Primary Bilateral Macronodular Adrenal Hyperplasia

Context: Primary bilateral macronodular adrenal hyperplasia (PBMAH) with glucose-dependent insulinotropic polypeptide (GIP)-dependent Cushing's syndrome is caused by ectopic expression of GIP receptor (GIPR) in the adrenal lesions. Such ectopic expression of GIPR was also reported in other endocrine neoplasm, notably in somatotroph pituitary adenomas from acromegalic patients with paradoxical increase of GH after oral glucose load, suggesting a common molecular pathogenesis. We aimed to identify the driver event responsible for GIP-dependent PBMAH with Cushing's syndrome and ectopic GIPR expression in somatotropinomas.
Methods: We conducted an international, multicenter, cohort study. We collected blood and adrenal samples from patients who had undergone unilateral or bilateral adrenalectomy for GIP-dependent PBMAH with Cushing's syndrome. Adrenal samples from patients with PBMAH and Cushing's syndrome without food-dependent cortisol production were used as controls. We further collected somatotropinoma specimens from acromegaly patients followed at two expert endocrine centers in France.
Results: 17 patients with familial or sporadic GIP-dependent PBMAH with Cushing's syndrome were studied. We identified germline heterozygous mutations in the lysine demethylase 1A (KDM1A) gene in all 17 patients. We further identified a recurrent deletion of the short arm of chromosome 1 harboring the KDM1A locus in the adrenal lesions of affected patients. None of the 25 patients in the control group had KDM1A germline or somatic alterations. Concomitant genetic inactivation of both KDM1A alleles resulted in loss of KDM1A expression in the adrenal lesions. RNA-sequencing revealed the global impact of KDM1A loss in adrenal tissue on gene transcription and identified differentially regulated genes including those encoding for GIPR and other G-Protein-Coupled Receptors that may be involved in adrenal tumorigenesis and regulation of steroidogenesis. In vitro pharmacologic inhibition, silencing and knock-out by CRISPR-Cas9 genome editing of KDM1A led to an increase in GIPR transcripts and protein in human adrenocortical H295R cells. Somatotropinoma samples from 78 patients with acromegaly were studied. 24% of these patients presented with a paradoxical rise of GH after oral glucose load and expressed ectopically GIP-receptor in their somatotropinoma. None of the somatotropinomas harbored KDM1A pathogenic variants, but those from patients with paradoxical GH response displayed a recurrent chromosome 1p loss.
Discussion: We identified germline inactivating KDM1A mutations and loss of heterozygosity as a genetic predisposition to GIP-dependent PBMAH with Cushing's syndrome following a tumor suppressor gene model of tumorigenesis. We currently perform genetic screening in first-degree relatives of patients with GIP-dependent PBMAH with Cushing's syndrome and clinical examination with biochemical testing in asymptomatic KDM1A variant carriers. We did not identify somatic KDM1A mutations in somatotropinomas expressing GIPR ectopically, however their recurrent 1p chromosome loss suggests that KDM1A haploinsufficiency may contribute to GIPR expression in those tumors.

Loss of KDM1A in Bilateral Macronodular Adrenal Hyperplasia With GIP-Dependent Cushing's Syndrome and in Acromegaly With Paradoxical GH Response to Oral Glucose

Adrenocortical carcinoma (ACC) is a rare aggressive cancer with heterogeneous behaviour. Disease surveillance relies on frequent imaging, which comes with significant radiation exposure. Here, we investigated the role of circulating cell-free DNA (ccfDNA)-related biomarkers for prognostication and monitoring of ACC.
We investigated 79 patients with ACC (29M/50F, 52±14yrs; 35 primary tumors [ACC-P] and 44 recurrences [ACC-R]); while 27 patients with adrenocortical adenomas [ACA] (9M/18F, 56±16yrs) and 19 healthy subjects (HS; 9M/10F, 37±9yrs) served as controls.
We extracted ccfDNA from 1-4 ml EDTA-plasma using Nonacus Cell3 Xtract or Qiagen QIAamp MinElute kit and quantified by fluorimeter (ccfDNA concentrations, Biomarker 1). Targeted next-generation sequencing (Illumina NextSeq500) was performed in a first subgroup of 52 baseline ccfDNA samples (23 ACC-P, 20 ACC-R, 8 ACA) using a customised panel of 30 ACC-specific genes (Cell3 Target Nonacus). Leucocyte DNA was sequenced to discriminate germline from somatic variants (Biomarker 2). Sequencing data from matched tumor DNA were available for 28 ACC (20 ACC-P, 8 R-ACC). A combined Biomarker score was calculated for prediction of clinical outcome. ACC-P had the highest ccfDNA concentrations (mean±SD 1.08±1.50 ng/µl) compared to ACC-R (0.29±0.23 ng/µl, P<0.05), ACA (0.17±0.13 ng/µl, P<0.005) and HS (0.11±0.07 ng/µl, P<0.005). Using a cutoff of 0.204 (median HS+2SD), 68% of ACC-P were postitive for Biomarker 1 (vs. ACC-R 55%, ACA 28%, HS 5%; P<0.0001 by Chi square test). At ccfDNA sequencing, 43% of ACC-P showed at least one somatic mutation (=positive Biomarker 2), vs. 15% in ACC-R and 0% in ACA. Mutational status at ccfDNA level matched with tumor DNA in 80% of cases. In 23 ACC-P with available sequencing data, the combined Biomarker score was strongly associated with both progression-free and overall survival (P<0.0001, HR 8.56, 95%CI 2.55-58.7, and P=0.0008, HR 13.3, 95%CI 3.77-47.1, respectively).
10 ACC-P were followed up for at least 6 months: 6 patients tumor-free at last CT scan were negative for Biomarker 1 whereas 3 out of 4 patients with early disease relapse were positive. In two recurrent cases, somatic mutations in ACC driver genes, i.e. MEN1 and ZNRF3, observed at baseline in both tumour and ccfDNA, remained detectable during monitoring.
In conclusion, ccfDNA-related biomarkers are frequently detected in patients with primary ACC. They may represent a promising, non-invasive tool to predict early disease progression and complement imaging in disease surveillance. Our findings on ccfDNA-based liquid biopsy will be validated in a larger cohort of ACCs with long-term follow up.

Circulating cell-free DNA-based biomarkers for prognostication and disease surveillance in adrenocortical carcinoma

Adrenocortical carcinoma (ACC) is an aggressive cancer with a significant risk of recurrence and high mortality rates. ACC has limited response to currently used treatments and there is an unmet clinical need to identify new treatment options.  Disease rarity coupled with absence of preclinical models of disease heterogeneity contributed to limited therapeutic progress in ACC. We previously established new in vitro and in vivo ACC models (CUACC1, CUACC2, CUACC9) with variable genomic alterations commonly seen in ACC tumors. Prior work demonstrated that maternal leucine embryonic kinase (MELK) is an oncogenic kinase in ACC, and that it can be targeted with the MELK inhibitor OTSSP167. To further evaluate the anti-tumor efficacy and mode of action of OTSSP167, this study used genetically diverse ACC cell lines (CUACC1, CUACC2, H295R), patient derived xenografts (PDX) (CUACC1 and CUACC9) and a cell line xenograft model (H295R) of ACC. ACC PDXs treated with 10 mg/kg OTSSP167 had significantly reduced MELK expression, tumor growth inhibition of more than 80%, reduced mitosis, and increased tumor tissue necrosis. Transcriptomic and phospho-proteomic analysis at multiple treatment timepoints in in vitro models revealed that OTSSP167 induced wide transcriptomic and proteomic changes involving effectors of signal transduction, cell cycle, protein translation, and epigenetic regulation to exert its anti-tumorigenic effects. Evaluation of OTSSP167's role in protein synthesis via a puromycin based SUnSET (surface sensing of translation) and kinase assay established that OTSSP167 inhibits de-novo protein synthesis, and targets RSK1 and its substrate rpS6 (S235/S236). Varying the degree of MELK silencing in H295R cell lines using a doxycycline inducible shRNA construct, inhibited RSK1 phosphorylation at T573 and S380 indicating a MELK-RSK1 axis in ACC. Additionally, OTSSP167's effect on the DNA damage pathway was validated, which activated the G2/M checkpoint protein Wee1, increased inhibitory phosphorylation of CDK1, causing an accumulation of cyclin B1 and G2/M arrest independent of TP53 status. Combination treatment with the Wee1 inhibitor AZD1775 and OTSSP167 enhanced OTSSP167 mediated cytotoxic effects in in-vitro models with an increase in mitotic entry and caspase mediated apoptosis. In summary, OTSSP167 exerted its anti-tumorigenic effects via inhibition of the protein translation machinery, DNA damage and cell cycle dysregulation causing drug sensitization. Results from these studies are a first demonstration of the anti-cancer effects of OTSSP167 in sensitization to Wee1 inhibition in ACC. These data suggest that OTSSP167 is a potential therapeutic option for patients with ACC and could be combined with Wee1 inhibitors in a clinical trial using a synthetic lethal approach.

Comprehensive Profiling of the MELK Inhibitor OTSSP167's Action and Its Effect on Sensitization to Wee1 Inhibitor AZD1775 in a Spectrum of ACC Models

Introduction: Complete Androgen Insensitivity Syndrome (CAIS) is characterized by a complete external genitalia appearance and testicular development in 46,XY individuals carrying inactivating mutations in the AR gene. Prophylactic gonadectomy versus saving gonads has been debatable in CAIS management, mainly due to the absence of an accurate biomarker for testicular germ cell tumors (GCT).Objective: to cross the preoperative pelvic MRI data with the histological gonadal findings from CAIS patients who underwent prophylactic gonadectomy to identify predictive factors of GCT development.Methods: We evaluated 30 gonads from 15 CAIS patients with proven AR mutation. All performed pelvic MRI before gonadectomy through the same MRI machine and were included in the MRI data analysis. Three blinded radiologists specialized in urology performed image analysis. The immunohistochemical markers PLAP, c-KIT, OCT3/4, SALL4, ER, alpha-inhibin, CD99, and calretinin were performed.Results: Nine (60%) performed gonadectomy after puberty (>16 years old). We identified one case of classical seminoma and one case of Sertoli cell neoplasia (at 18 and 19 years of age, respectively). Overall, the prevalence of neoplasia was 13.3%.  No case of premalignant intratubular germ cell neoplasia was detected.Histological data revealed a high prevalence of benign lesions (n=12; 80%). Leydig Cell Hyperplasia (67%), stromal fibrosis (53%), Sertoli Cell nodules (47%), hamartomatoums nodules (40%) and paratesticular cysts (67%) which are more frequent after puberty (p<.01). MRI detected heterogeneity in all cases harboring hamartomatoums nodules, Sertoli cell nodules, and testicular neoplasia. Benign lesions were bilateral in all cases. Regarding the MRI data, the presence of testicular cystic mass (by tumoral necrosis) on MRI and tumor size (higher among malignancies) correlated with malignancy (p=.008 and p=.03, respectively), whereas gonadal location (inguinal/abdominal), presence of solid nodules, T2WI and postcontrast features, nodule diffusion restriction, and presence of perigonadal cysts did not. Chronological age >16 years was associated with malignancy with a 4.3 OR (1.6 – 11.69).Conclusion: Pelvic MRI is sensitive to detect gonadal nodules in CAIS patients, regardless of the gonad position, but most nodules are benign. Testicular malignancy should be suspicious in the presence of testicular cystic mass and large testis size in CAIS patients >16 years old.

Testicular Magnetic Resonance Imaging (MRI) Findings and Predictive Factors of Gonadal Neoplasia in Complete Androgen Insensitivity Syndrome

BackgroundDisorders/Differences of sex development (DSD) may be associated with adverse psychosocial and psychosexual outcomes in adults. However, there is a paucity of information on health-related quality of life (QoL) outcomes in parents and children with DSD and a lack of instruments available for evaluating these outcomes. Recently, this has led to the development of Parent Self-Report and Proxy-Report QoL questionnaires (QoL-DSD), validated measures for parents of young children with DSD, comprising 63 items within 13 domains and 25 items within 5 domains, respectively.ObjectiveWhile retaining the original domain structure of the QoL-DSD questionnaires, we aimed to develop short forms of the QoL-DSD, optimizing their use in routine clinic settings.MethodsShort forms of the DSD-QoL Parent Self-Report (QoL-DSD Short PSR) and Parent Proxy-Report (QoL-DSD Short PPR) questionnaires were developed following exploratory factor analysis with maximum likelihood and varimax rotation, using previous QoL-DSD data from 132 parents. Long and resulting short form questionnaires were completed online by 18 parents of children with DSD, under 7 years of age, attending endocrine and urology clinics at one tertiary hospital in Scotland.ResultsItem selection for the short forms — QoL-DSD Short PSR and QoL-DSD Short PPR — based on item factor loadings of >0.8, produced questionnaires containing 16 and 7 items, respectively. Eighteen parents completed both long and short forms of the Parent Self-Report for children aged <7 years, and a subset of these (n=13) also completed long and short forms of the Parent Proxy-Report for children aged 2 to 7 years. Of the 18 target children, all were boys with a median age of 3.6 years (range 0.4, 6.6) and 10 (59%) had proximal hypospadias. Overall, agreement was achieved between the short and long questionnaires in 9 out of 12 (75%) domains on the Parent Self-Report and 4 out of 5 (80%) domains on the Parent Proxy-Report. Correlations between total scores on the short and long forms were 0.508 to 0.897 in 9 of 12 domains in the QoL-DSD Short PSR. Parental feedback (n=18) regarding the acceptability of the short versus long forms was evaluated using a 5-point Likert score: 83% (versus 66%) of parents agreed the length of time (less than 3 minutes) taken to complete short forms was acceptable, 39% preferred the short forms compared with 11% whom preferred the longer version, 45% (versus 22%) stated a preference to complete the short forms should they be implemented routinely at clinic visits in the future.Conclusions Short forms of the QoL-DSD for parents of young children with DSD may be more acceptable for use in a routine outpatient setting to evaluate psychosocial distress experienced by young children with DSD and their caregivers. Further psychometric validation in a larger cohort is warranted.

Development and Validation of a Short Version of the Quality of Life DSD Questionnaire (QoL-DSD) for Parents of Young Children with Disorders/Differences of Sex Development

Background: Hypospadias is a common human congenital anomaly occurring in ~1: 200 to 1: 300 live male births in the USA, with an unexplained doubling in incidence over previous decades. Historically, 46XY Differences of Sex Development (DSD) patients, with severe hypospadias have lacked a genetic diagnosis.  With the advent of commercially available DSD genomic panels, this is now changing (GeneDX. Neonatal 46 2021, In-vitae 2021).  The goal of this study is to review the anatomical characteristics of severe hypospadias that can accurately define patients that have 46XY DSD and to determine the practical utility of performing a genomic workup in these rare 46XY DSD patients with severe hypospadias.Patients: 14 patients seen in disorders of sexual development clinic between 2018 – 2020 with severe hypospadias defined by an ectopic urethral meatus typically in the scrotum or perineum, an abnormally small glans penis size (<14 mm at maximal width in the first year of life), severe curvature and associated foreskin fusion or penoscrotal transposition were enrolled in the study.Methods: Patients who met the criteria for 46XY disorder of sexual development and severe hypospadias were offered a genomic work-up in consultation with pediatric genetics. Patients were identified through chart review. Data extracted included karyotype, hypospadias phenotype including stretched penile length at diagnosis, age at genetic diagnosis/genomic workup, gene defect/ pathologic variant, gender identity, and clinical course.Main Outcome Measurements: The main outcome measured was the presence of a genetic mutation that can be linked to the disorder of sexual development. Secondary outcomes measured included gender identity, clinical course, and histological findings if a biopsy was obtained.Results: Of the 14 patients that underwent genomic analysis, 8 had documented pathologic variants known to cause hypospadias. Pathologic genetic variants include defects in the AR (3), SRD5A2 (1), NR5A1 (2), WT1 (1), and ARTX (1).  Five had no evidence of pathologic variants based on the Invitae and GeneDx commercial 46XY DSD panel (see Table 2 for genes tested).  One had a genetic variant of unknown significance, FREM2. The patient with WT1 was consequently found to have a Wilms tumor and the patients with SRD5A2 are now undergoing adrenal insufficiency surveillance after their genetic testing.Conclusions: Patients with 46XY DSD and severe hypospadias can benefit from genetic evaluation as their underlying disorders may unveil mutations that could have potentially life-altering consequences and change surveillance and monitoring.

Utility of Genomic work-up for 46XY Disorder of Sex Development Patients with Severe Hypospadias

Study Objective:This case series was designed to describe cases of polycythemia among transgender male adolescent patients undergoing treatment with testosterone.Methods:Retrospective data was collected for our Pediatric Gender Clinic between Jan 2014 and June 2021. Variables assessed included demographic and laboratory data, presence of comorbidities, testosterone dose and level at the time of polycythemia, concurrent treatment with other hormonal treatments, and duration of therapy. Polycythemia in children was defined as hematocrit (HCT) +2 SD above mean for age and gender. Male reference range was used for HCT after 3 months of treatment.Results:Seven of 131 patients were identified with polycythemia (5.3%). The mean age was 17.9 ± 3.7 (SD) years at the time of polycythemia and 6 of 7 patients were overweight or obese. All patients were receiving testosterone by injection. The mean testosterone dose was 51.4 ± 13.3 (SD) mg/m²/2wks or 92.9 mg/2wk ±22.1 (SD). All patients developed polycythemia developed at doses below 75 mg/m²/2wks. The mean testosterone level at the time of polycythemia was 588 ± 315.3. (SD) ng/dL and the mean length of treatment before polycythemia occurred was 18.6 ± 6.2 (SD) months; the shortest duration of treatment was 10 months. Two patients have received concurrent treatment with progestin therapy. Obstructive sleep apnea (OSA) was identified in 1 patient.Conclusion:Polycythemia occurred in 5.3% of our transgender male patients receiving testosterone therapy. This is a known complication of testosterone therapy that may appear with a relatively low dose and after a relatively short duration of treatment. Patient 1Patient 2Patient 3Patient 4*Patient 5Patient 6Patient 7Age at time of polycythemia, years19.116.616.721.816.917.616.6BMI at time of polycythemia, kg/m²25.239.634.130.949.619.426.7Smoking/vaping NNNVapingNNNComorbidities: Asthma, OSANAsthma, mild OSAAsthmaNNNNAdditional hormonal therapyNMedroxyprogesterone acetate injectionEtonogestrel implantNNNNHCT level at baseline, %46.544.143.050.444.245.841.1HCT level at the time of polycythemia, %
55.050.150.053.050.550.650.8Testosterone level at the time of polycythemia, ng/dL3997033455254821253416Length of Tx with testosterone, months10141426182523Dose of testosterone, mg/2wk801008060130100100Dose of testosterone, mg/m²/2wk47.369.741.231.249.664.556.5Table 1. Characteristics of patients with polycythemia.* Patient was taking an injectable dose of testosterone for 6-8 months prior to the first visit to our clinic.

Case Series of Transmasculine Adolescents Who Developed Polycythemia on Gender Affirming Hormone Therapy.

Objective: GnRH agonists (GnRHa), used to prevent development of undesired secondary sex characteristics for gender diverse youth (GDY), may impede pubertal bone mineral accrual increasing the risk for osteopenia/osteoporosis during adulthood. Based on findings of low bone mineral density (BMD) scores (relative to natal sex) prior to GnRHa [1], we hypothesized BMD might be associated with body mass index (BMI) in GDY.Method: To test our hypothesis, we reviewed medical records for 27 GDY; 16 were designated female at birth (DFAB) and 11 were designated male at birth (DMAB). Age at presentation, gender identity, ages at initiation of GnRHa and gender affirming hormone treatment (GAHT), duration of GnRHa treatment prior to GAHT initiation, auxologic parameters including BMI, and lumbar spine BMD obtained from GE lunar IDXa dual-energy X-ray absorptiometry (DEXA) were extracted. Lumbar spine BMD was used for statistical analysis. Statistical analysis was performed with Microsoft Excel Analysis ToolPak. A linear regression model was used to determine significance of BMI and GnRHa as predictors of BMD Z-score using a significance level of 0.05. Legal gender marker was used to determine BMD Z-scores.Results: BMI ranged from 16.93 kg/m² to 35.79 kg/m² (5th to the 99th percentile). DEXA scan results were available for 27 GDY with age range 10-25 years; mean age (±SD) was 17±3 years. BMD of the lumbar spine was obtained from 0-61 months (mean 30 ± 21 months) following initiation of GnRHa. Length of GnRHa treatment prior to GAHT initiation ranged from 0-53 months (mean 14 ± 12 months).Individuals were at different stages of the gender affirmation process including 1) No medical treatments, 2) GnRH agonist only, 3) GAHT only, and 4) combined GnRH agonist and GAHT. Using a linear regression model, length of GnRHa therapy and length of GnRHa without GAHT were not significantly associated with lumbar spine BMD in our sample (p = 0.44 and 0.29, respectively). Using a linear regression model, BMI was a significant positive predictor of higher lumbar bone mineral density (p = 0.03).Conclusion: Sex hormone exposure during adolescence and young adulthood is essential for bone health. While GnRH agonists lower endogenous sex hormone exposure, in our sample, lower BMD was not associated with total duration of GnRHa therapy or duration of GnRHa treatment prior to GAHT. Rather BMD was positively associated with BMI regardless of gender affirming medical therapies. Limitations include small sample size, retrospective design, and the use of legal gender marker for normative values. The next step will be larger, prospective studies to identify predictors of low BMD in GD youth. This information will guide future management regarding BMD monitoring.References: 1. Lee JY,et al., J Endocr Soc. 2020;4(9): bvaa065.

The developmental origin and the specification of the adrenal cortex in humans and cynomolgus monkeys

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WNT2B Deficiency Results in Hyperreninemic Hypoaldosteronism in Humans and Mice

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