Discovering cellular programs of intrinsic and extrinsic drivers of metabolic traits using LipocyteProfiler.

A primary obstacle in translating genetic associations with disease into therapeutic strategies is elucidating the cellular programs affected by genetic risk variants and effector genes. Here, we introduce LipocyteProfiler, a cardiometabolic-disease-oriented high-content image-based profiling tool that enables evaluation of thousands of morphological and cellular profiles that can be systematically linked to genes and genetic variants relevant to cardiometabolic disease. We show that LipocyteProfiler allows surveillance of diverse cellular programs by generating rich context- and process-specific cellular profiles across hepatocyte and adipocyte cell-state transitions. We use LipocyteProfiler to identify known and novel cellular mechanisms altered by polygenic risk of metabolic disease, including insulin resistance, fat distribution, and the polygenic contribution to lipodystrophy. LipocyteProfiler paves the way for large-scale forward and reverse deep phenotypic profiling in lipocytes and provides a framework for the unbiased identification of causal relationships between genetic variants and cellular programs relevant to human disease.

Retrospective review of changes in testosterone dosing and physiologic parameters in transgender and gender-diverse individuals following hysterectomy with and without oophorectomy.

BACKGROUND: As more transgender and gender-diverse patients undergo hysterectomy, gaps in knowledge remain about how testosterone dosing or other physiologic parameters change following surgery and how these are influenced by concomitant oophorectomy. AIM: The aims of this study were to determine the incidence of testosterone dosing change after gender-affirming hysterectomy and to compare this incidence between patients who underwent oophorectomy and ovarian preservation. METHODS: This multicenter retrospective cohort study consisted of transmasculine patients who underwent hysterectomy for gender affirmation. OUTCOMES: Outcome measures included testosterone dosing changes at least 3 months following hysterectomy, as identified by clinical documentation, as well as clinical and laboratory parameters assessed for a change after hysterectomy: free and total testosterone, estradiol, hemoglobin, hematocrit, total cholesterol, weight, and blood pressure. RESULTS: Of the 50 patients, 32 (64%) underwent bilateral oophorectomy, 10 (20%) unilateral oophorectomy, and 8 (16%) maintained both ovaries. Eight percent (n = 4) changed testosterone dosing following hysterectomy. Those who underwent bilateral oophorectomy were no more likely to change their testosterone dose than those who did not (P = .09). Those who also used menstrual suppression were 1.31 times more likely to change doses of testosterone after hysterectomy (95% CI, 1.09-1.82; P = .003). For those who had pre- and posthysterectomy laboratory and clinical values, the majority saw no clinically significant change. However, among patients who underwent bilateral oophorectomy, the calculated free testosterone increased by 90.1 ± 288.4 ng/dL (mean ± SD), and estradiol dropped by 20.2 ± 29.0 pg/mL. CLINICAL IMPLICATIONS: In a field where access to care can be a significant barrier, there is unlikely to be a need for routine reassessment of testosterone dose or laboratory parameters following hysterectomy, whether or not a bilateral oophorectomy occurs. STRENGTHS AND LIMITATIONS: Limitations of the study include its retrospective nature and the lack of consistent clinical laboratory testing, which resulted in limited data about any given hormonal change. The heterogeneity of our population limited the number of patients undergoing or not undergoing oophorectomy; however, it allowed our study to more truly reflect a clinical environment. CONCLUSION: In a multisite cohort of individuals who underwent hysterectomy for gender affirmation, few patients changed testosterone dosing after surgery. In addition, dosing change was not associated with the presence or absence of bilateral oophorectomy, and most measured laboratory values remained consistent following hysterectomy.

Retrospective Review of Sexual and Reproductive Health Conversations During Initial Visits of Adolescents Seeking Gender-Affirming Testosterone.

STUDY OBJECTIVE: To use a retrospective review of sexual and reproductive health (SRH) counseling that occurred during initial visits of adolescents seeking testosterone gender-affirming hormone therapy to determine the feasibility of using such visits to manage SRH DESIGN: Retrospective chart review SETTING: Children's hospital, multidisciplinary gender clinic PARTICIPANTS: Transgender male and nonbinary patients assigned female at birth (TGD-M) aged 15-17 seen for initiation of testosterone between January 1, 2010, and December 31, 2019 INTERVENTIONS: Not applicable MAIN OUTCOME MEASURE(S): Counseling on (1) testosterone impact on fertility and (2) fertility preservation; assessment of (3) desire for gender-affirming surgery, (4) sexual activity, (5) sexual orientation, and (6) human papilloma virus vaccination as documented during the initial visit. RESULTS: Of 195 patients who met the inclusion criteria, only 3 (1.5%) had all 6 measures addressed. The median number addressed was 4 out of 6 (IQR = 2-5/6), with fertility counseling (95.9%, n = 187) being most common, followed by assessment of surgery desire (74.4%, n = 145), sexual orientation (69.2%, n = 135), and sexual activity (69.2%, n = 135). The odds of being asked about sexual orientation were 5.3 times higher in patients who endorsed sexual activity than in those who did not (P < .001; 95% CI, 9.8-10.3). CONCLUSION: Providers of adolescent gender-affirming hormone therapy regularly assess and counsel on certain aspects of SRH as part of their initial visits for those seeking testosterone. Our data suggest that these initial visits for patients seeking testosterone represent an opportunity to expand SRH assessment and counseling among TGD-M adolescents.

Transcriptome variation in human tissues revealed by long-read sequencing.

Regulation of transcript structure generates transcript diversity and plays an important role in human disease1-7. The advent of long-read sequencing technologies offers the opportunity to study the role of genetic variation in transcript structure8-16. In this Article, we present a large human long-read RNA-seq dataset using the Oxford Nanopore Technologies platform from 88 samples from Genotype-Tissue Expression (GTEx) tissues and cell lines, complementing the GTEx resource. We identified just over 70,000 novel transcripts for annotated genes, and validated the protein expression of 10% of novel transcripts. We developed a new computational package, LORALS, to analyse the genetic effects of rare and common variants on the transcriptome by allele-specific analysis of long reads. We characterized allele-specific expression and transcript structure events, providing new insights into the specific transcript alterations caused by common and rare genetic variants and highlighting the resolution gained from long-read data. We were able to perturb the transcript structure upon knockdown of PTBP1, an RNA binding protein that mediates splicing, thereby finding genetic regulatory effects that are modified by the cellular environment. Finally, we used this dataset to enhance variant interpretation and study rare variants leading to aberrant splicing patterns.

Family Building Perspectives of Assigned Female at Birth Transgender and Gender Diverse Adolescents Seeking Testosterone Gender-Affirming Hormone Therapy.

Purpose: The purpose of this study was to assess the future family building desires of assigned female at birth (AFAB) transgender and gender diverse (TGD) adolescents initiating hormone therapy, and to characterize the individuals interested in adoption. Methods: This was a retrospective chart review of AFAB TGD adolescents ages 15-17 years old initiating testosterone gender-affirming hormone therapy between 2010 and 2019, analyzing interest in adoption, demographics, and gender-affirming care. Results: Of 195 AFAB TGD adolescents asked about family planning goals, 58% (n = 113) indicated desire for adoption in their future, and 13.3% (n = 26) had no desire for children. There was no difference between those who did and did not want to adopt in terms of age at time of first visit (p = 0.22), or race distribution (p = 0.45); however, straight-identified patients were more likely to desire adoption (p = 0.02) than people with other sexual orientations. Fifty-nine percent (n = 110) of those who did not have a history of adoption and/or experience with the child welfare system desired adoption, compared with 22% (n = 2) of those with a history (odds ratio, 5.14; 95% confidence interval, 1.04-25.39; p = 0.05). Conclusion: Some AFAB TGD adolescents endorse adoption as their desired pathway to parenthood. Clinicians should be sensitive to the complexities of parenthood desires of AFAB TGD patients and have resources to direct patients to more information. Further research is needed to better understand why many AFAB TGD adolescents desire adoption, how this changes with age, and the barriers they face in achieving their goals.

A tripartite complex of suPAR, APOL1 risk variants and αvß3 integrin on podocytes mediates chronic kidney disease.

Soluble urokinase plasminogen activator receptor (suPAR) independently predicts chronic kidney disease (CKD) incidence and progression. Apolipoprotein L1 (APOL1) gene variants G1 and G2, but not the reference allele (G0), are associated with an increased risk of CKD in individuals of recent African ancestry. Here we show in two large, unrelated cohorts that decline in kidney function associated with APOL1 risk variants was dependent on plasma suPAR levels: APOL1-related risk was attenuated in patients with lower suPAR, and strengthened in those with higher suPAR levels. Mechanistically, surface plasmon resonance studies identified high-affinity interactions between suPAR, APOL1 and αvß3 integrin, whereby APOL1 protein variants G1 and G2 exhibited higher affinity for suPAR-activated avb3 integrin than APOL1 G0. APOL1 G1 or G2 augments αvß3 integrin activation and causes proteinuria in mice in a suPAR-dependent manner. The synergy of circulating factor suPAR and APOL1 G1 or G2 on αvß3 integrin activation is a mechanism for CKD.

Dynamin Autonomously Regulates Podocyte Focal Adhesion Maturation.

Rho family GTPases, the prototypical members of which are Cdc42, Rac1, and RhoA, are molecular switches best known for regulating the actin cytoskeleton. In addition to the canonical small GTPases, the large GTPase dynamin has been implicated in regulating the actin cytoskeleton via direct dynamin-actin interactions. The physiologic role of dynamin in regulating the actin cytoskeleton has been linked to the maintenance of the kidney filtration barrier. Additionally, the small molecule Bis-T-23, which promotes actin-dependent dynamin oligomerization and thus, increases actin polymerization, improved renal health in diverse models of CKD, implicating dynamin as a potential therapeutic target for the treatment of CKD. Here, we show that treating cultured mouse podocytes with Bis-T-23 promoted stress fiber formation and focal adhesion maturation in a dynamin-dependent manner. Furthermore, Bis-T-23 induced the formation of focal adhesions and stress fibers in cells in which the RhoA signaling pathway was downregulated by multiple experimental approaches. Our study suggests that dynamin regulates focal adhesion maturation by a mechanism parallel to and synergistic with the RhoA signaling pathway. Identification of dynamin as one of the essential and autonomous regulators of focal adhesion maturation suggests a molecular mechanism that underlies the beneficial effect of Bis-T-23 on podocyte physiology.