United States

Objective: Primary Bilateral Macronodular Adrenal Hyperplasia (PBMAH) is a heterogeneous disease characterized by adrenal macronodules and variable levels of cortisol excess, with not clearly established clinical diagnostic criteria. PBMAH can be caused by germline inactivating variants of the tumor suppressor gene ARMC5. We aimed to better characterize ARMC5-mutated and wild-type patients and to identify predictive criteria for ARMC5 variants.
Methods: We included 352 consecutive index patients from 12 European centers, sequenced for germline ARMC5alteration because of PBMAH suspicion, regardless their clinical presentation. Clinical, biological and imaging data were collected retrospectively. Sensitivity, specificity, negative and positive predictive values for the prediction of ARMC5variant were calculated for various parameters.
Results: 53 patients (15.1%) carried 40 different ARMC5 germline pathogenic variants and showed a more distinct phenotype than non-mutated patients for cortisol excess (24-hour urinary free cortisol 2.32 vs. 1.11-fold ULN, respectively, p&lt;0.001; plasma cortisol after 1 mg dexamethasone suppression test 337.5 vs. 142.4 nmol/L, respectively, p&lt;0.001) and adrenal morphology (maximal adrenal diameter 104 vs. 83 mm, respectively, p&lt;0.001; 9.8 vs. 3.2 adrenal nodules, respectively, p&lt;0.001), with more frequent metabolic complications such as diabetes (51.0 vs. 35.4%, respectively, p=0.038) and hypertension (88.0 vs. 70.6%, respectively, p=0.014). Thus, ARMC5-mutated patients were more often surgically or medically treated in order to control cortisol excess (67.9 vs. 36.8%, respectively, p&lt;0.001). Among operated patients, a bilateral adrenalectomy was more often performed in those carrying an ARMC5 pathogenic variant than in wild-type patients (56.7 vs. 32.3%, respectively, p=0.019). Even if all patients were more often primarily investigated in front of adrenal incidentaloma than clinical evidence for Cushing&#39;s syndrome (73 vs. 27%, respectively), ARMC5 patients were more often referred for Cushing&#39;s syndrome than wild-type patients (44 vs. 24%, respectively, p=0.004). To improve the ARMC5 mutation rate, the association of a clear bilateral adrenal involvement with evidence for autonomous cortisol secretion (defined at least by a plasma cortisol after 1 mg dexamethasone suppression test above 50 nmol/L) holds the better yield with a 27% specificity and a 20% positive predictive value, and a 100% sensitivity and negative predictive value, meaning that a useless genotyping could have been avoided for more than 20% of negative patients, without missing any mutated patient.
Conclusion: We report the largest series of index case patients investigated for ARMC5 with clinical characterization and confirm that ARMC5 pathogenic variants are associated with a more severe phenotype. In order to minimize negative ARMC5 screening, genotyping could be limited to clear bilateral adrenal involvement on imaging and autonomous cortisol secretion, with an optimum yield for routine clinical practice. These findings will also help better define PBMAH diagnostic criteria.

ENDO 2022

Identification of Predictive Criteria for the Primary Bilateral Macronodular Adrenal Hyperplasia Gene ARMC5: A European Series of 352 Unselected Patients.

**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. 

Objective: Primary Bilateral Macronodular Adrenal Hyperplasia (PBMAH) is a heterogeneous disease characterized by adrenal macronodules and variable levels of cortisol excess, with not clearly established clinical diagnostic criteria. PBMAH can be caused by germline inactivating variants of the tumor suppressor gene ARMC5. We aimed to better characterize ARMC5-mutated and wild-type patients and to identify predictive criteria for ARMC5 variants.
Methods: We included 352 consecutive index patients from 12 European centers, sequenced for germline ARMC5alteration because of PBMAH suspicion, regardless their clinical presentation. Clinical, biological and imaging data were collected retrospectively. Sensitivity, specificity, negative and positive predictive values for the prediction of ARMC5variant were calculated for various parameters.
Results: 53 patients (15.1%) carried 40 different ARMC5 germline pathogenic variants and showed a more distinct phenotype than non-mutated patients for cortisol excess (24-hour urinary free cortisol 2.32 vs. 1.11-fold ULN, respectively, p<0.001; plasma cortisol after 1 mg dexamethasone suppression test 337.5 vs. 142.4 nmol/L, respectively, p<0.001) and adrenal morphology (maximal adrenal diameter 104 vs. 83 mm, respectively, p<0.001; 9.8 vs. 3.2 adrenal nodules, respectively, p<0.001), with more frequent metabolic complications such as diabetes (51.0 vs. 35.4%, respectively, p=0.038) and hypertension (88.0 vs. 70.6%, respectively, p=0.014). Thus, ARMC5-mutated patients were more often surgically or medically treated in order to control cortisol excess (67.9 vs. 36.8%, respectively, p<0.001). Among operated patients, a bilateral adrenalectomy was more often performed in those carrying an ARMC5 pathogenic variant than in wild-type patients (56.7 vs. 32.3%, respectively, p=0.019). Even if all patients were more often primarily investigated in front of adrenal incidentaloma than clinical evidence for Cushing's syndrome (73 vs. 27%, respectively), ARMC5 patients were more often referred for Cushing's syndrome than wild-type patients (44 vs. 24%, respectively, p=0.004). To improve the ARMC5 mutation rate, the association of a clear bilateral adrenal involvement with evidence for autonomous cortisol secretion (defined at least by a plasma cortisol after 1 mg dexamethasone suppression test above 50 nmol/L) holds the better yield with a 27% specificity and a 20% positive predictive value, and a 100% sensitivity and negative predictive value, meaning that a useless genotyping could have been avoided for more than 20% of negative patients, without missing any mutated patient.
Conclusion: We report the largest series of index case patients investigated for ARMC5 with clinical characterization and confirm that ARMC5 pathogenic variants are associated with a more severe phenotype. In order to minimize negative ARMC5 screening, genotyping could be limited to clear bilateral adrenal involvement on imaging and autonomous cortisol secretion, with an optimum yield for routine clinical practice. These findings will also help better define PBMAH diagnostic criteria.

technical paper

Background: Patients with adrenal hormone excess demonstrate an increased cardiovascular risk and mortality. We aimed to determine the impact of adrenal hormone excess on the metabolic profile that included plasma lipoproteins, total branched-chain amino acids (BCAA), ketone bodies, and GlycA (a proinflammatory glycoprotein biomarker). Method: We conducted a single-center cross-sectional study of consecutive patients with adrenal adenomas, Cushing syndrome (CS), and pheochromocytoma/paragangliomas (PPGL) between January 2015 and May 2021. Adrenal adenomas comprised nonfunctioning adenomas (NFA), adenomas with mild autonomous cortisol secretion (MACS), and primary aldosteronism (PA). Malignant and other benign tumors were excluded. Collected fasting plasma samples were measured using nuclear magnetic resonance spectroscopy for lipoproteins, BCCA, ketone bodies and GlycA. The results were compared to referent subjects from PREVEND (Prevention of Renal and Vascular End-stage Disease) study. Lipoprotein Insulin Resistance Index (LP-IR) which predicts incident diabetes was calculated based on 6 lipoprotein parameters. Results: NFA (n=166, median age 60.1years, 63.3% women), MACS (n=158 (median age 61.8 years, 62. 0% women), CS (n=101, median age 43.8 years, 85.1% women), PA (n=71 (median age 55.8 years, 31. 0% women), PPGL (n=43, median age 55.5 years, 46.5% women) was diagnosed in 539 patients. Referent subjects (n=6540) were younger with a median age of 48.4, 50.2% women. When compared to referent subjects, and after age and sex-adjustment, GlycA was highest in patients with CS (OR 3.3, 95% CI 2.8-3.8), followed by PPGL (OR 2.3, 95%CI 1.8-2.9), MACS (OR 2, 95%CI 1.7-2.3), NFA (OR 1.9, 95%CI 1.7-2.2), and PA (OR 1.5, 95%CI 1.2-1.8). When compared to referent subjects, and after sex- and age adjustment, all patient groups demonstrated increase in BCAA: CS (OR 2.7 (95%CI 2.3-3.2), PPGL (OR 2.3, 95% CI 1.8-2.9), NFA (OR 2.4, 95% CI 2.1-2.7), PA (OR 2. 0, 95%CI 1.7-2.5), MACS (OR 2.2, 95% CI 1.7-2.6). Total ketone bodies were increased in patients with cortisol excess (OR of 1.4, 95%CI 1.2-1.5 in CS, and OR of 1.2, 95%CI 1-1.3 in MACS) and NFA (OR 1.2, 95%CI 1.1-1.3), but not in PA or PPGL. All patient groups except PPGL were more likely to have a higher LP-IR score. When compared to referent subjects, the highest sex- and age-adjusted LP-IR increase was in patients with CS (OR 1.9 (95%CI 1.5-2.2), followed by MACS (OR 1.4, 95% CI 1.2-1.7), PA (OR 1.4, 95%CI 1.1-1.8), and NFA (OR 1.4, 95% CI 1.2-1.7). Conclusion: Patient with adrenal adenomas and PPGL demonstrate an increase in GlycA, BCAA, and ketone bodies - biomarkers associated with adverse cardiometabolic disorders and mortality. All patients except those with PPGL also demonstrated a higher LP-IR index that was reported to predict incident diabetes. Patients with NFA demonstrated an adverse metabolic profile similar to patients with MACS.

Metabolic Profiling in Adrenal Tumors Demonstrates Enhanced Chronic Inflammation, Branched Chain Amino Acids and Ketone Bodies, Biomarkers that Predict High Cardiometabolic Risk

Background: Pheochromocytoma (Pheo)/Paraganglioma (PGL), together PPGL, are rare but life-threatening tumors, with tumor relapse (TR) rates after initial surgery of 16.5%, and genetic mutations in about 30-40% of patients. Long-term follow-up (FU) and genetic testing in all patients are recommended. Questions remain, however, as to the most suitable duration of FU, the prognostic value of genetic diagnoses and of clinical, biochemical or radiological findings during FU, of the incidence of TR after recommended 10 years FU of low-risk sporadic Pheos, or of TR on morbidity and mortality.
Objective, patients, and methods: We retrospectively determined the overall survival (OAS), progression-free survival (PFS) and tumor-recurrence (TR) in 173 patients with PPGL initially operated and followed-up at a single tertiary referral center from 1988 to 2020 by Kaplan-Meier Estimates, and assessed age, sex, hormonal activity, tumor size, and metastatic spread at first diagnosis, pathohistological PASS score, mutation-positive PPGLs (MP-PPGL), TR, and comorbidities (associated with increased cardiovascular risk) as to independent prognostic markers thereof by multivariate Cox-regression. Certificats of the Austrian death registry were also obtained.
Results: In 8 (5%) PPGLs there was no FU in the charts, but death certified occurring 65±28 (mean±SD, median 2.7) months after first diagnosis. Mean (±SD) age of the remaining 165 (43.9% female) patients (94.5 Pheos, 3.0% multiple PGLs, 2.5% head-and-neck PGLs) was 49(±16) years, mean (range) FU 90(3-537) months. 93 (54%) patients underwent genetic testing, 37 (40%, 21% of entire cohort) had MP-PPGL (mean±SD age 33.8±13.2 yrs, OAS 391 months). 5 of these 37 (13.5%), and 7 of 57 (12%) mutation-negative (MN-PPGL, mean±SD age 53.3±14.8 years, p<0.01 vs. MP-PPGL, OAS 190 months) died during FU (p=0.03). Male sex, higher age, presence of comorbidities, and primary metastatic disease were independent negative prognostic factors for OAS by univariate, but only age and metastatic disease remained significant by multivariate analysis. As to PFS, higher age and no hormonal activity were negatively associated by univariate, but not by multivariate analysis, as were all other tested parameters.
Conclusions: The cohort of our study is the third-largest original report on long-term FU and prognostic markers in PPGL. While others (Ayola-Ramirez et al, JECM 2011) in 371 patients found tumor size and sympathetic PGL independently associated with OAS, we did not. Patients may have harbored more aggressive tumors in that study, as higher primary metastatic spread (25% of Pheos, 60% of sympathetic PGL) was identified. We identified only age and metastatic disease as independent predictors of OAS, in line with data from Korea (Kim J H, et al., Endocrinol Metab. [Seoul]) in 1048 patients (9% with primary metastatis) and with a metaanalysis comprising 703 PPGLs (Crona J, Endocr Relat Cancer, 2019).

Long-term Follow-Up of Pheochromocytoma/Paraganglioma (PPGL) after first diagnosis: a retrospective single-center study of 173 patients

Tumor-induced osteomalacia (TIO) is a rare paraneoplastic syndrome wherein small tumors secrete excess fibroblast growth factor 23 (FGF23) leading to hypophosphatemia, osteomalacia, bone pain, fractures, and muscle weakness. Burosumab is an anti-FGF23 monoclonal antibody approved for treatment of TIO. We report end of study (EOS) data from the open-label phase 2 UX023T-CL201 trial (NCT02304367) of burosumab in adults with TIO.
In UX023T-CL201, participants received burosumab (0.3–2.0 mg/kg) by subcutaneous injection every 4 weeks for 48 weeks with an extension up to 300 weeks (~6 years). Co-primary endpoints were change in serum phosphorus at Week 24 and change in excess osteoid by trans-iliac crest bone biopsy at Week 48. Additional endpoints included changes in bone biomarkers, activity on radionucleotide bone scan representing fractures/pseudofractures, patient-reported outcomes, and safety measures.
This analysis included 14/17 UX023T-CL201 participants; excluding those with other diagnoses (n=3).
Mean (SD) serum phosphorus improved from 1.6 (0.5) mg/dL at baseline to 2.3 (0.5) mg/dL when averaged across the mid-point of the dose interval through Week 24 (co-primary endpoint). Levels further improved (2.6 [0.4] mg/dL; n=7) to within the normal range (2.5–3.6 mg/dL) at Week 240. Most measures of osteomalacia improved at Week 48 (co-primary endpoint), including osteoid/bone volume, osteoid thickness, and mineralization lag time. However, osteoid surface/bone surface showed no change.
Bone biomarkers (P1NP, CTx, BALP) followed similar trends. Upon initiation of burosumab, markers briefly increased by Week 24 as bone healing began, then gradually decreased with further treatment, reaching normal (P1NP, CTx) or near-normal (BALP) ranges by Week 240.
At baseline, 249 areas of increased radionucleotide uptake indicating fracture activity were detected across 14 participants. By Week 144, 36% (72/201) and 13% (26/201) regions of abnormal uptake were fully or partially restored, respectively. By EOS, further reduction of active fractures and fewer new areas of uptake were observed. New fractures at Week 144 were observed in a single participant (considered a burosumab partial responder) and between Week 144 and EOS in another participant unrelated to burosumab (car accident).
Participants reported sustained mean reduction from baseline pain at Week 240 per Brief Pain Inventory and significantly per SF-36 Bodily Pain score. Significant reductions in baseline fatigue at Week 240 were reported per Brief Fatigue Inventory and SF-36 Vitality scores. SF-36 physical health (Role Physical, Bodily Pain, Component Score) and mental health (Social Function, Vitality) significantly improved from baseline at Week 240.
The observed safety profile of long-term burosumab in participants with TIO was consistent with the known safety profile of burosumab and similar to patients with XLH.
Long-term burosumab therapy was associated with improved serum phosphorus, osteomalacia, bone turnover, fracture incidence and healing, health-related quality of life, and reductions in pain and fatigue in adults with TIO.

Long-Term Burosumab Therapy Provides Sustained Benefit in Patients with Tumor-Induced Osteomalacia: End of Study Findings From the Pivotal Phase 2 Study

X-linked hypophosphatemia (XLH) is a rare, heritable disorder wherein excess FGF23 leads to renal phosphate wasting and impaired activation of vitamin D. XLH is characterized by rickets and osteomalacia leading to bone pain, skeletal deformities, and short stature. The XLH Disease Monitoring Program (DMP; NCT03651505) is a prospective, multinational outcomes study of the longitudinal progression of clinical, radiographic, and biochemical features of XLH in children and adults. We assessed the impact of the recombinant human IgG1 monoclonal antibody to FGF23, burosumab, on measures of bone health in children with XLH at Year 1 of the DMP.
As of March 2021, 287 children (<18 years) had completed the Year 1 visit. Participants were grouped by when they first received burosumab: Group 1, prior to DMP enrollment (N=68; mean (SD) age 9.5 (3.6) years); Group 2, during the DMP (N=150; 8.7 (5.0) years); Group 3, never, but receiving conventional therapy (N=69; 8.8 (5.3) years).
Before receiving burosumab, serum phosphorus levels were below normal; after burosumab initiation, serum phosphorus in Groups 1 and 2 rapidly normalized, with sustained improvements up to 3 years of treatment. Serum phosphorus remained low in Group 3. Before burosumab, serum alkaline phosphatase levels were above normal; with continued burosumab therapy, mean levels gradually declined towards normal and were maintained for up to 4 years.
The Radiographic Global Impression of Change (RGI-C) assessed rickets and lower limb deformity from DMP baseline to Year 1, with positive values denoting improvement. Mean (SD) RGI-C rickets global scores were +1.0 (0.9) for Group 1 (n=44), +1.5 (0.7) for Group 2 (n=39), and +0.3 (1.2) for Group 3 (n=3). Lower limb deformity RGI-C scores were +0.3 (0.6) for Group 1 (n=42), +0.4 (0.8) for Group 2 (n=39), and +0.8 (1.1) for Group 3 (n=3). Height Z-score change from DMP baseline at Year 1 was 0.1 (0.3) for Group 1 (n=58), 0.1 (0.4) for Group 2 (n=90), and -0.8 (3.1) for Group 3 (n=18). There were no new safety concerns or reports of hyperphosphatemia.
These real-world data affirm that normalization of serum phosphorus is rapid and sustained following burosumab initiation, resulting in improved serum alkaline phosphatase levels which positively impact bone mineralization. Improvements in rickets, growth, and lower limb deformity occur in the first year of burosumab treatment and continue in those who started burosumab prior to entering the DMP. The absence of new safety concerns or reports of hyperphosphatemia up to 4 years after burosumab initiation is also notable. The 10-year DMP study design will provide understanding of long-term effects in these patients with continued burosumab therapy.

Characterizing the Impact of Burosumab on Bone Health in Children with X-Linked Hypophosphatemia: Results from Year 1 of the Disease Monitoring Program

Introduction: X-linked hypophosphatemia (XLH), a dominant disorder caused by disease-associated variants in the PHEX gene, affects males and females of all ages. Rickets and osteomalacia may be present along with short stature, lower limb deformity, muscle pain and/or weakness/fatigue, bone pain, fractures and poor fracture healing, joint pain/stiffness, hearing difficulty, enthesopathy, osteoarthritis, and dental abscesses. Patients with XLH have below-normal serum phosphate and elevated serum FGF23. The Hypophosphatemia Gene Panel Sponsored Program provides a no-charge genetic test to patients  to confirm a clinical XLH diagnosis or to aid in the diagnosis of suspected genetic hypophosphatemia.
Methods: Individuals aged ≥6 months with either a clinical XLH diagnosis or suspicion of genetic hypophosphatemia, as evidenced by 2 or more clinical signs/ symptoms, were eligible for testing. The next generation sequencing panel includes 17 genes: ALPL, CLCN5, CTNS, CYP2R1, CYP27B1, DMP1, ENPP1, FAH, FAM20C, FGF23, FGFR1, GNAS, OCRL, PHEX, SLC34A1, SLC34A3, and VDR. Copy number variant (CNV) detection was also performed. Variants were interpreted according to the joint consensus from the American College of Medical Genetics and Genomics and the Association for Molecular Pathology using Invitae's proprietary point-based scoring system, Sherloc. In all analyses, data were summarized using descriptive statistics.
Results: One thousand six hundred sixty-eight unrelated individuals, ages 6 months to 87 years (mean = 27.4±21.2 years, median = 22.5 years), were tested between 27 February 2019 and 23 October 2021. The majority of individuals were female (66.6%) and of self-reported White ancestry (54.9%).
Eight hundred seventy-nine (52.7.%) unrelated individuals had a PHEX variant: 825 (93.9%) were either pathogenic or likely pathogenic (P/LP) and 54 (6.1%) were variants of uncertain significance (VUS). Of the 789 (47.3%) cases where no PHEX P/LP or VUS was found, 116 (14.7%) had molecular diagnoses associated with other genes/disorders: ALPL (71 with 1 variant, 12 with 2 variants), FGF23 (8), CYP27B1 (14), SLC34A3 (5), and two or fewer molecular diagnoses for DMP, CTNS, ENPP1, and OCRL.
Four hundred and twenty-eight unique P/LP PHEX variants were detected: 136 deletions, duplications or insertions (<100 bp); 41 copy number variants (>100 bp); 74 splice-site variants; 177 single nucleotide variants. Additional family member testing/clinical information/observation of variants resulted in 25 VUS being reclassified to P/LP (impacting 50 individuals), highlighting the value of cascade family testing/clinical information to resolve VUS. Notably, the frequency of PHEX VUS has decreased from 8.9% in 2019 (41 weeks) to 4.4% in 2021 (44 weeks).
Conclusions: The frequency of any PHEX variant classified as VUS has decreased more than two-fold since 2019. RNA analyses to resolve VUS may further improve molecular diagnostic yield. Program results provide a high molecular diagnostic yield for XLH and other genetic hypophosphatemia disorders and new insight into XLH-associated PHEX variants.

Hypophosphatemia Gene Panel Sponsored Program: A High Yield of Molecular Diagnoses from Clinically Confirmed XLH and Suspected Genetic Hypophosphatemia

Background: 2% of youth identify as transgender. Many have puberty blocked with gonadotropin-releasing hormone analogues (GnRHa), followed by testosterone (T) or estradiol (E2). Bone mineral density (BMD) effects of these therapies are understudied.
Objective: For youth on GnRHa alone or T or E2 +/- GnRHa, to evaluate: (1) BMD z-scores; (2) BMD z-score differences for those +/-GnRHa; and (3) associations between GnRHa duration and BMD z-score.
Methods: Cross-sectional study of transgender youth (n=56 age 10.4-19.8 years, 53% female sex assigned at birth) undergoing total body dual-energy X-ray absorptiometry (Hologic Discovery A). Data are presented as median and interquartile range, group differences evaluated with Mann Whitney U tests and associations with Spearman correlations.
Results: BMD z-scores on GnRHa alone (n=19) were -0.5 (-1.4, 0.3) using male norms and -0.8 (-1.7, 0.3) using female norms. BMD z-score for transgender males (TM) on T (n=21, 5 with past GnRHa): -0.2 (-0.6, 0.0) using male norms, 0.4 (-0.3, 0.8) using female norms. BMD z-score for transgender females (TF) on E2 (n=16, 6 on current/past GnRHa): -0.4 (-1.2, 0.3) using male norms, -0.2 (-1.0, 0.7) using female norms. TM on T with prior GnRHa had significantly lower BMD z-scores than TM on T alone (p=0.004); but no differences for TF on E2 +/- GnRHa. GnRHa duration was inversely correlated with BMD z-score (male norms: r=-0.5, p=0.005, female norms: r=-0.4, p=0.029). BMD z-score was unrelated to length of T/E2 therapy or sex steroid concentrations.
Conclusions: Longer durations of GnRHa therapy were associated with worse BMD z-scores.

Bone Mineral Density in Transgender Youth on Gender Affirming Therapies

Background: Cytochrome P450c17 (17α-hydroxylase, encoded by CYP17A1) and cytochrome P450c11 (11β-hydroxylase, encoded by CYP11B1) are essential for production of adrenal cortisol. The expression of CYP17A1 and CYP11B1 in adrenocortical cells is stimulated by adrenocorticotropic hormone (ACTH) through the cAMP and protein kinase A signal transduction pathway. Several growth factors can act as autocrine/paracrine regulators for modulating the steroidogenesis in the adrenal cortex. Among these factors, bone morphogenetic protein-4 (BMP4), a member of the superfamily of transforming growth factor-beta (TGFβ) has been previously reported to have an inhibitory effect on adrenal cell CYP17A1 expression and dehydroepiandrosterone (DHEA) production [1]. The role of BMP4 within the adrenal and its mechanisms regulating steroidogenesis are still unclear. In the present study, we examined human adrenal expression of BMP4 and its chronic effects on the H295R adrenal cell line. Objective: To define the autocrine/paracrine role of BMP4 in adrenocortical zonation and adrenal steroidogenesis. Methods: BMP4 expression in normal human adrenal glands was studied using microarray analysis with laser-captured adrenal capsule and cortical zones. Adrenal BMP4 protein expression was examined following immunohistochemistry (IHC) with a rabbit polyclonal antibody against human BMP4. In vitro studies were performed using H295R and human adrenal cells treated with or without ACTH (10nM), forskolin (10 µM) and BMP4 (50 ng/mL). The experimental medium was collected every 24 h and analyzed for cortisol. Experiments were terminated at 96 h followed by isolation of RNA and qPCR analysis for PPIA (normalization transcript), cytochrome P450 cholesterol side-chain cleavage (CYP11A1), CYP17A1 and CYP11B1. Results: Microarray analysis of adrenal zone RNA demonstrated a BMP4 expression gradient with zona glomerulosa (ZG) expressing 3 and 11-fold higher levels than the zona fasciculata (ZF) and zona reticularis (ZR), respectively. IHC confirmed that BMP4 expression was highest in the ZG and the outer ZF. Administration of BMP4 to cultures of H295R cells caused a significant decrease in basal and forskolin-stimulated cortisol production at 48 h. By 96 h, BMP4 inhibited forskolin-stimulated cortisol by 60%. Cortisol inhibition occurred in conjunction with an inhibition in forskolin-stimulated CYP17A1 (↓75%) and CYP11B1 (↓65%). However, CYP11A1 expression was unaffected. In primary cultures of human adrenal cells BMP4 inhibited the ability of ACTH to increase cortisol production (↓75%), as well as CYP11B1(↓65%) and CYP17A1 (↓57%) expression. Conclusion: Our findings indicate that the human adrenal has outer cortex localized expression of BMP4 that could play a paracrine/autocrine role in zone-specific production of aldosterone vs. cortisol through BMP4 selective inhibition of CYP17A1 and CYP11B1. Bibliography: 1. Rege J, Nishimoto HK, Nishimoto K, Rodgers RJ, Auchus RJ, Rainey WE. Bone Morphogenetic Protein-4 (BMP4): A Paracrine Regulator of Human Adrenal C19 Steroid Synthesis. Endocrinology 2015; 156: 2530–2540.

Bone Morphogenetic Protein-4 (BMP4) Inhibits Adrenal Cell Cortisol Synthesis by Blocking CYP17A1 and CYP11B1 Expression.

BackgroundCystic fibrosis-related diabetes (CFRD) is a unique type of diabetes that is associated with significantly increased morbidity and mortality in both children and adults with cystic fibrosis (CF). The prevalence of CFRD progressively increases with age such that more than half of adults with CF develop CFRD. Early diagnosis and treatment are associated with improvements in body weight and pulmonary function, reduction in the frequency of pulmonary exacerbations, and improved overall survival. Since patients with CFRD may present with no symptoms, screening is recommended starting from the age of 10 years with an annual oral glucose tolerance test (OGTT). Especially during the COVID-19 pandemic, requiring an in-person clinic visit can be challenging, which may lead to a delayed diagnosis of CFRD.ObjectivesThe purpose of this project was to develop a state-of-the-art technique to detect changes in glucose levels of patients with CF by developing a deep learning-based audio classification tool. Preliminary work by our group suggested that voice characteristics could distinguish between patients with CFRD patients and patients with CF but without CFRD. We hypothesize that high blood glucose levels may cause laryngeal soft tissue swelling leading to changes in voice characteristics.MethodsWe performed a prospective cross-sectional study in adult patients with CF recruited from Emory CF Clinic from March to December 2021. We recorded 5-second voice samples of a sustained /a/ vowel via a portable digital microphone. The spectrogram was extracted via the Mel frequency cepstral coefficient. The training to test the dataset ratio was 80: 20. 20% of the training dataset were randomly selected to serve as a validation dataset. We designed a convolutional neural network (CNN) architecture for CFRD patients' voice classification.ResultsThere were a total of 100 subjects consisting of 43 patients with CFRD and 57 patients with CF without diabetes. The male to female ratio was approximately 60: 40 in both groups. Patients with CFRD had similar mean age and mean BMI to patients without CFRD. There was a significantly higher point of care glucose level in CFRD patients. The mean duration of a CFRD diagnosis was 9 years and the mean HbA1c level was 7.26 in the CFRD group. The performance of the VGG model CNN classifier achieved 98.7% and 94.92% accuracy on training and validation datasets, respectively. On the test dataset, the model achieved 73.53% sensitivity 69.77% specificity and 71.43% accuracy.ConclusionsWe found a deep learning-based audio screening tool for CFRD could be potentially used as an alternative tool for screening in the CF community. A convolutional neural network algorithm demonstrated high sensitivity and specificity to adequately differentiate between patients with and without CFRD. Larger prospective studies are required to test this technology in patients with every form of diabetes.

Deep learning-based voice screening technique for cystic fibrosis related diabetes

Background: The link between birth-size, early childhood growth and later life cardiovascular (CV) risk is well recognised. The contribution to CV risk of growth and cell differentiation during pre-implantation embryo development is unknown.
Aims: Develop a transcriptomic model of developmental trajectory in the pre-implantation embryo and link this model to birthweight, child growth and CV risk in later life.
Methods: Single cell RNA velocity¹ and pseudotime analyses were used to infer developmental trajectory in the preimplantation human embryo². Time series clustering was used to identify genes with similar activity patterns. These data were compared with independent genomic data from four further data sets. I) Birthweight genome wide association study (GWAS)³. II) Genes correlated with gestation⁴. III)  A previously identified set of 55 genes that classify pre-hypertensive young adults using their blood methylome at age 0, 7 and 17 years⁵. IV) A set of genes with differential methylation associated with linear childhood growth⁶. Data sets III) and IV) were derived from the Avon Longitudinal Study of Parents and Children (n=6487). Enrichment was assessed using the hypergeometric test and confirmed via random permutation (1x10⁵-fold) and z-score analysis. Random Forest was used to determine the predictive value presented as the area under the curve (AUC) of the receiver operating characteristic.
Results: 8256 genes contributed to the velocity of mRNA change in the preimplantation embryo and a temporal wave pattern was revealed with peaks of velocity at 3, 4, and 5-days post-fertilisation. Genes associated with the velocity of the developmental trajectory formed 6 clusters, in which metabolic pathways were over-represented (adj. p<0.02).
We found that 16/60 birthweight GWAS (I) and 3096/10521 gestation correlated genes (II) were associated with embryonic developmental trajectory (both 2-fold enrichment, p<5x10-3). Furthermore, 11/55 pre-hypertensive associated genes (III) were linked with embryonic developmental trajectory (>10-fold enrichment, p<1x10-8), these 11 genes predicted adult pre-hypertension in small for gestational age catch-up growth (cuSGA) with an AUC=0.98. Of the 8256 developmental trajectory genes, 307 were differentially methylated in linear childhood growth (IV) (2.3-fold over-enrichment, p<1x10-8).
Conclusion: Waves of gene expression velocity occur in preimplantation human embryos. These waves mark major developmental transitions and form a transcriptomic developmental trajectory which is associated with both birthweight and gestation. The transcriptomic developmental trajectory genes are over-enriched for genes associated with the linear phase of childhood growth and for genes associated with and predictive for the development of adult pre-hypertension in cuSGA. Pre-implantation gene expression can therefore be linked to the origins of adult disease.
1. LaManno, et al. (2018). Nature. 560(7719): 494-8.
2. Petropoulos, et al. (2016). Cell. 165(4): 1012-26.
3. Horikoshi, et al. (2016). Nature 538(7624): 248-252.
4. Rasmussen, et al. (2022). Nature. https://doi.org/10.1038/s41586-021-04249-w
5. Garner, et al (2021). JES. 5: A654–A655.
6. Garner, et al (2019) ESPE Abstracts 92FC12.6

Preimplantation embryo development is associated with both birth size and post-natal growth and can predict adult cardiovascular risk

Introduction: Early-onset obesity is a cardinal feature of rare genetic obesity disorders. According to the Endocrine Society guideline, genetic screening is indicated in selected cases with age of onset (AoO) of severe obesity (grade ≥2) <5 years. However, this cut-off is not validated.
Aims: To present the detailed BMI characteristics of children and adolescents with rare genetic obesity disorders; to evaluate whether the following growth chart characteristics can aid in assessing which children should be screened for genetic obesity disorders: AoO of obesity, AoO of severe obesity, BMI standard deviation scores (SDS) at yearly age intervals.
Methods: In this prospective observational study, children with non-syndromic and syndromic genetic obesity disorders treated at our tertiary obesity center were included. Growth measurements from birth onwards were collected. Children with obesity from a population-based cohort study with follow-up until age 10 years were included as an unselected reference cohort. Diagnostic performance (sensitivity [sens], specificity [spec], positive likelihood ratio [LR+], area-under-the-curve [AUC]) for AoO of obesity and severe obesity and for BMI-SDS at yearly age intervals was calculated.
Results: We included 64 children with genetic obesity disorders (32 non-syndromic, 32 syndromic) and 298 control children with obesity. At intake, median age of children with genetic obesity was 10.5 years (IQR 7.0–14.7) and mean BMI-SDS +3.1 ± 1.1. Median AoO of obesity was 1.2 years (IQR 0.6–3.7) in non-syndromic genetic obesity, 2.1 years (IQR 0.9–4.2) in syndromic genetic obesity, and 3.8 years (IQR 2.3–6.2) in the control population. For non-syndromic genetic obesity, optimal cut-off value for AoO of obesity was ≤1.5 years: sens 0.60, spec 0.88, LR+ 5.22, AUC 0.79 (p<0.001). For syndromic genetic obesity, optimal cut-off was ≤3.0 years: sens 0.68, spec 0.68, LR+ 2.06, AUC 0.67 (p=0.001). AoO of severe obesity showed worse diagnostic performance than AoO of obesity in both non-syndromic (AUC 0.58, p=0.20) and syndromic genetic obesity (AUC 0.58, p=0.21). Moreover, when the guideline cut-off <5 years was used, AoO of severe obesity showed a negative diagnostic performance (non-syndromic genetic obesity: sens 0.76, spec 0.13, LR+ 0.88; syndromic genetic obesity: sens 0.85, spec 0.14, LR+ 0.98). BMI-SDS showed good diagnostic performance for non-syndromic genetic obesity across the age intervals (AUCs 0.79-0.89, all P<0.001) but not for syndromic genetic obesity (AUCs 0.54-0.71, P-values ranging from <0.001-0.50).
Conclusion: This study shows that growth charts characteristics such as BMI-SDS and AoO of obesity (grade 1), but not AoO of severe obesity (grade ≥2), could be useful to distinguish between children with genetic obesity disorders and children with obesity from a population-based cohort study. However, all investigated growth charts characteristics showed misclassification, especially in syndromic genetic obesity, indicating that additional clinical features should be present to warrant genetic testing in these children.

Identification of Predictive Criteria for the Primary Bilateral Macronodular Adrenal Hyperplasia Gene ARMC5: A European Series of 352 Unselected Patients.

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