United States

Background and Objectives: Human fertility is attained following puberty due to finely orchestrated events driven by hypothalamic release of the excitatory neuropeptide neurokinin B (NKB), which in turn stimulates the release of kisspeptin and then GnRH to activate the downstream pituitary-gonadal axis. Our group has identified that loss-of-function mutations in Makorin Ring Finger Protein 3 (MKRN3) cause central precocious puberty (CPP). Mkrn3 is highly expressed in the hypothalamus of both male and female mice in early postnatal life, then declines prior to the onset of puberty. Mutations in MKRN3 E3 ubiquitin ligase domain, identified in patients with CPP, result in reduced auto-ubiquitination. However, the mechanisms of action of MKRN3 and its targets remain largely unknown. We hypothesized that MKRN3 inhibits the reproductive axis through interactions with hypothalamic substrates, targeting them to degradation pathways.
Methods and Results: Using interactome analysis and candidate approaches in vitro, we identified poly(A) binding protein cytoplasmic 1 and 4 (PABPC1 and 4) and insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) as MKRN3 interactors, consistent with reports from other research groups. Additionally, our proteomic analysis revealed that Igf2bp1 levels are increased in the hypothalamus of Mkrn3 knockout (KO) mice versus wild type animals. Remarkably, we found that MKRN3-IGF2BP1 interaction is mediated by RNA, as their interaction was abrogated by RNase treatment. However, an MKRN3 missense mutation associated with CPP, p.C364F, within the E3 ubiquitin ligase domain, did not affect the RNA mediated interaction. Preliminary data from RNA co-IP, aiming to identify putative RNA targets, suggested that MKRN3 and IGF2BP1 bind to TAC3 mRNA. We further identified an increase in Nkb protein levels in the hypothalamus of Mkrn3 KO mice. We also showed that NKB is a substrate of MKRN3 E3 ubiquitin ligase activity in vitro by demonstrating increased ubiquitination of NKB in cells co-transfected with expression vectors encoding MKRN3 and NKB and treated with proteasome inhibitor. Additionally, MKRN3 overexpression resulted in degradation of NKB protein by western blot analysis, and alteration of NKB intracellular localization by immunofluorescence. These effects were impaired by MKRN3 missense mutations (p.C340G and p.C364F) in the E3 ubiquitin ligase motif, suggesting a pathophysiological mechanism of MKRN3 mutations in CPP.
Conclusions: We identified that MKRN3 interacts with PABPC1, PABPC4 and IGF2BP1 and targets NKB to degradation. We showed that NKB degradation was mediated by the MKRN3 E3 ubiquitin ligase domain, as mutations within this motif abrogated NKB degradation. However, this mutation did not affect MKRN3 RNA-mediated binding to IGF2BP1, highlighting that multiple domains of MKRN3 may contribute to its actions. Here, we propose a mechanism by which MKRN3, interacting with PABPCs and IGF2BP1 and targeting NKB for degradation, may act to inhibit the reproductive axis.

ENDO 2022

MKRN3 inhibits the reproductive axis by interacting with key hypothalamic substrates and targeting neurokinin B to degradation pathways.

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technical paper

Psychological stress is linked to infertility and reproductive dysfunction by suppressing the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator and pulsatile luteinising hormone (LH) secretion. The posterodorsal subnucleus of the medial amygdala (MePD) is a key upstream regulator of GnRH pulse generator activity and displays increased neuronal activation in response to psychological stressors. The MePD is primarily a GABAergic nucleus with a strong GABAergic projection to hypothalamic reproductive centres, however, their functional significance has not been determined. We hypothesis that MePD GABAergic signalling is a crucial mediator of psychological stress-induced suppression of pulsatile LH secretion. We used a chemogenetic approach to selectively silence MePD GABA neurones during psychological stress to determine the effect on pulsatile LH secretion. Female ovariectomised Vgat-cre mice (n=7) were virally infected to selectively express inhibitory hM4DGi-DREADDs in MePD GABA neurones. Mice were exposed to either predator odour (PO) or restraint stress and given an intraperitoneal injection of either saline or the DREADD chemical activator clozapine-N-oxide (CNO, 5mg/kg). Statistical significance was determined using a one-way ANOVA and post-hoc Tukey test. Data are presented as mean ± SEM and p<0.05 was considered significant. PO exposure significantly increased LH inter-pulse interval (IPI) in the saline group (prestress: 19.05 ± 1.16 min vs. PO: 29.28 ± 3.16 min, p<0.05, n=7) and this effect was blocked by DREADD-mediated inhibition of MePD GABA neurones (prestress: 20.59 ± 1.12 min vs. PO:  21.31 ± 1.76 min, p>0.05, n=7). Restraint stress dramatically increased LH IPI in the saline group (prestress: 17.78 ± 1.02 min vs. restraint: 45.71 ± vs 5.50 min, p<0.05, n=7) and DREADD-mediated inhibition of MePD GABA neurones blocked this effect (prestress: 18.57 ± 1.97 min vs. restraint: 27.26 ± 4.02 min, p>0.05, n=7). Direct projections from the MePD to the GnRH pulse generator in the hypothalamic arcuate nucleus (ARC) have been identified, however, their phenotype is unknown. In this study, we optogenetically stimulated potential MePD GABAergic projection neurone terminals in the ARC and determined the effect on LH IPI. MePD GABA neurones in female Vgat-cre ovariectomised mice (n=6) were virally infected to express channelrhodopsin 2 protein and MePD GABAergic terminals in the ARC were selectively stimulated by blue light via a chronically implanted fibre-optic cannula in the ARC. Sustained optogenetic stimulation at 10 and 20Hz of MePD GABAergic terminals in the ARC dose-dependently suppressed pulsatile LH secretion (LH IPI; control: 23.34 ± 2.11 min vs. 10Hz: 38.08 ± 3.61 min; 20Hz: 56.67 ± 3.33 min, p<0.05, n=6). These findings confirm a functionally significant MePD GABAergic projection to the ARC and highlight the importance of GABA signalling in the MePD in mediating stress-induced suppression of reproductive function. Collectively, these findings provide novel insight into the amygdala neural circuitry underpinning stress-induced reproductive function.

GABAergic Signalling in the Posterodorsal Medial Amygdala Mediates Psychological Stress-induced Suppression of the GnRH Pulse Generator in Female Mice

In female animals, ovarian estradiol (E2) acts as negative feedback inhibition of GnRH secretion and positive feedback stimulation at ovulation. Both E2-regulated mechanisms work via stimulation or repression of distinct neuronal populations of Kisspeptin (KP)-synthesizing neurons. While E2 clearly stimulates AVPV KP neurons to increase kiss1 expression during the preovulatory surge, mechanisms required for optimal potentiation of maximal GnRH surge release remain incompletely characterized. We used two immortalized KP cell lines demonstrating increased kiss1 expression under high E2 (KTaV-3), or kiss1 suppression under low E2 (KTaR-1), to probe GnRH receptor (GnRHR) expression levels under E2 conditions of varying doses and times of exposure. Additionally, we have begun to investigate transcriptional mechanisms underlying gnrhr expression in KP neurons in vitro.
AVPV-derived KTaV-3 cells were treated with either E2 alone (5-100pM) or E2 with progesterone (4-40nM) for varying durations (4-96h), either constitutively or under modulating levels approximating changes found during the murine estrous cycle. All treatments were undertaken in serum-free (SF) media (DMEM) following a 50% serum synchronization. After total RNA isolation, cDNAs were probed with mouse gnrhr primers. Results reveal that gnrhr expression, not observed in typical growth media conditions, is induced in KTaV-3 cells to varying degrees dependent on prior E2/P4 exposure. Following shorter treatment times (12-24h), proestrus levels (100pM E2; 4nM P4) of sex steroids noticeably induced gnrhr, while exposure of KTaV-3 cells to a 96h estrous cycle revealed maximal gnrhr expression at the diestrus II→proestrus transition, with exposure cycles concluding with declining E2/rising P4 exhibiting no to minimal gnrhr expression. These results suggest that maximal KP neuronal expression of gnrhr may occur prior to the E2 peak on proestrus, potentially preparing AVPV KP neurons for GnRH surge release 12-16h later. Similar steroid conditions also increased dlx3 expression, a transcription factor required for GnRHR in pituitary, only in KTaV-3 cells. In KTaR-1 cells, gnrhr expression was observed only following E2 decreases, while dlx3 was constitutively elevated. Since cFos/JunB complex formation on the gnrhr promoter has been implicated in gonadotrope GnRHR levels, we explored how transfection of these transcription factors might affect gnrhr expression in KTaV-3 cells. Preliminary results reveal that fos/jun co-transfection stimulates gnrhr expression maximally at intermediate (DII) E2 levels, while higher E2 levels are required with fos or jun expression alone. While reciprocal GnRH-Kisspeptin connections have not yet been observed in vivo, these results suggest KP neurons may possess the capacity to respond to intrahypothalamic GnRH only in certain estrous cycle stages, by differentially modulating GnRH receptivity in the AVPV. These findings supplement the prevailing model of feed-forward KP regulation of GnRH with potential short-loop KP-GnRH positive feedback regulation, providing an increased understanding of feedback mechanisms required for normal neuroendocrine regulation of reproduction.

Immortalized AVPV Kisspeptin Neurons Transiently Express GnRH Receptors under Specific Sex Steroid Conditions in vitro.

Introduction: The Ames strain, a spontaneous mutant mouse of the Prop1 gene, is deficient in GH, TSH, PRL and gonadotropins. In recent years, it has been the focus of studies on aging and there is a growing interest in elucidating the role of hormone replacement (HR) in the hypothalamic-pituitary-gonadal (HPG) axis. Aim: The present study aimed to characterize sexual maturation and fertility restoration in isogenic strain under or not HR. Material and methods Five homozygous male animals were treated via intraperitoneal injections, starting 30 days postnatally, with levothyroxine 3x/week and recombinant human GH 5x/week for 40 days, followed by maintenance applications of both hormones once a week until they complete 90 days post natal. The sexual maturity was evaluated looking for balanoprepucial opening and descendent testis comparing untreated homozygous (n=5) with their wild type siblings (n=5). Fertility was evaluated by mating known fertile wild type animals with treated and untreated  animals for 8 times. Reproductive parameters were evaluated between groups using histological sections of the testes (HE) and classified according to johnsen score where 1-2 was classified as absent germline cells and 8-10 normal development. At the sacrifice, testicular weight was measured using scale and spermogram was done looking for motility and viability in the sperm collected in the seminiferous tube. Gata2 transcription factor and pituitary hormones Lh, Fsh, Tsh, Prl and Gh transcriptional analysis were performed by RT-qPCR. Results: The homozygous treated animals presented a 2 weeks delay in the age of sexual maturation compared to wild animals. Their fertility and reproductive parameters were restored noticed by increased testicular weight, improved spermatogenesis, similar morphology of seminiferous tubules (johnsen score 8.7) and spermogram compared to the wild type, besides presenting offspring when mated to their wild siblings. Sexual maturity was absent in most of the untreated homozygous animals presenting no offspring. The reproductive parameters in the untreated homozygous animals presented reduced testicular weight, size of seminiferous tubes leading to johnsen score as 5 and an azospermic spermogram in all animals. Interestingly 2 untreated homozugous animal had spontaneous maturation. Gata2 was significant decreased in the untreated animal (0,49 ± 0,19) compared to wild type (1 ± 0,15) (p≤ 0.019). LH transcriptional pattern was significant increase in the animals under treatment (1.56 ± 0.68) compared to homozygotes without hormonal intervention (0.36 ± 0.34) (p≤ 0.048). Conclusion: Ames mutant mice under treatment with GH and levothyroxine replacement reached sexual maturation and restored fertility and the mechanics behind this phenomenon will be explored using RNA seq in the future.

Hormone replacement promotes sexual maturation and fertility restoration in mice with congenital hypopituitarism harboring Prop1 mutation

BACKGROUND: Hypoactive Sexual Desire Disorder (HSDD) is associated with dysfunctional brain activation in regions governing sexual responses, resulting in a deficiency or absence of sexual desire with marked distress. It is of major clinical importance given it affects 8% of men with detrimental effects on quality of life, interpersonal relationships and fertility, but so far has no licensed treatment options. The reproductive neuropeptide kisspeptin offers a putative therapeutic target owing to its emerging role in modulating reproductive behaviour in animal models and healthy men. However, there are no studies examining its effects in HSDD. To address this, we performed the first clinical study of kisspeptin in men with HSDD.
METHODS: We examined the effects of kisspeptin administration (vs placebo) on brain activity during short and long erotic video tasks using functional MRI in 32 men with HSDD (mean ±SEM age 37.9 ±1.5 y, BMI 24.9 ±1.0 kg/m²). The short video task used 20-second segments of erotic video with non-erotic video as control. During the long video task, participants viewed a continuous eight-minute erotic video. To provide functional and behavioural relevance for the associated fMRI brain responses during the long erotic video, simultaneous penile tumescence and subjective level of arousal were recorded. Participants also completed psychometric and behavioural questionnaires. Standard analysis methods were used for fMRI data from the short videos task, and the long videos task used regressors derived from the subjective arousal and penile tumescence data. The statistical threshold used for both was Z=2.3, p < 0.05 (cluster-corrected).
RESULTS: In response to visual erotic stimuli, kisspeptin administration significantly increased penile tumescence during the long video task compared to placebo, with kisspeptin increasing penile tumescence by 56% at six-minutes (p=0.002). In addition, kisspeptin increased participant-reported happiness about sex (p=0.02). During both video tasks, kisspeptin significantly modulated brain activity compared to placebo in key structures of the sexual-processing network, providing a mechanistic pathway for the increases in physiological and behavioural measures. In response to short erotic videos, kisspeptin enhanced left middle frontal gyrus and left anterior cingulate activity, and decreased activity in bilateral parahippocampus (all p<0.05). During the long video task, kisspeptin enhanced right fusiform gyrus and bilateral visual cortex activity, and decreased left frontal pole, right posterior cingulate and bilateral precuneus activity (all <0.05). Additionally, we observed positive correlations between the effects of kisspeptin on aforementioned brain activity and psychometric parameters of sexual desire and arousal (all p<0.01).
CONCLUSION: Collectively, we demonstrate for the first time that kisspeptin administration in men with HSDD increases penile tumescence and psychometric measures of sexual desire and arousal by modulating sexual brain processing. Taken together, our data suggest that kisspeptin-based therapeutics may offer a novel, effective and much-needed clinical strategy for men with HSDD.

Kisspeptin Increases Penile Tumescence and Sexual Brain Processing in Men with Hypoactive Sexual Desire Disorder

Background and Objective: Makorin Ring Finger Protein 3 (MKRN3), an inhibitor of the KISS1 and TAC3 promoters in the hypothalamic arcuate nucleus (ARC), is an important player in age of menarche and etiology of central precocious puberty. However, its potential role in delayed puberty and/or hypogonadism is not defined. Mkrn3 expression in the ARC declines prior to the onset of puberty and remains low in adulthood, supporting its role as an inhibitor of pubertal onset. Neonatal hypothalamic overexpression of Mkrn3 leads to delayed puberty in female mice; this study aimed to explore the effects of MKRN3 overexpression in the postpubertal mouse. We hypothesized that MKRN3 overexpression in the ARC of postpubertal female mice would result in suppression of the HPG axis, manifested as hypogonadotropic hypogonadism. Methods and Results: Adult female C57BL/6 mice were stereotaxically injected into the ARC with a recombinant adeno-associated virus (rAAV-Mkrn3-IRES-EGFP;n=12) to overexpress Mkrn3, or with a control virus (rAAV-EGFP;n=9). Daily vaginal smears collected six weeks post-injection showed that rAAV-Mkrn3 injected mice spent significantly more time (62.7%) in diestrus compared to controls (41.3%;P<0.01). Mean body weight did not statistically differ between groups. Eight weeks after viral injection, gonad-intact females underwent stimulation tests while in diestrus. Tail vein blood samples were collected at baseline and 20, 40 and 60 minutes after stimulation. In response to the NK3R agonist, senktide, evaluated at two doses (5 nmol and 10 nmol ip, respectively), Mkrn3-overexpressing mice had a reduced peak (i.e., T20) LH response compared to controls (P<0.001 for both dosing paradigms). In contrast, in response to kisspeptin-10 (1 nmol ip), mice responded with similar peak serum LH levels. Subsequently, tests were repeated seven days after ovariectomy and estradiol capsule replacement (OVX+E2). Peak LH levels (T20) were again significantly lower in response to senktide (5 nmol ip) in the rAAV-Mkrn3 injected females (P<0.001). Peak serum LH levels (T20) were not different in response to kisspeptin-10 (1 nmol) or GnRH (1.25 ng/g body weight). Additionally, there were no significant differences in serum LH levels between groups 7 days after OVX or OVX + E2. Conclusions: In this novel mouse model, developed to investigate the mechanism of action of MKRN3, an important neuroendocrine controller of puberty initiation, these findings suggest that MKRN3 can exert inhibitory effects on the reproductive axis in female mice beyond the juvenile period. In these mice, response to kisspeptin and GnRH was preserved but the response to senktide was blunted. These findings suggest that Mkrn3 may act in ARC kisspeptin neurons to inhibit kisspeptin synthesis or secretion and/or potentially the action of neurokinin B via its receptor. Modulation of MKRN3 expression may be a future therapeutic target for reproductive disorders.

The Impact of Makorin Ring Finger Protein 3 (MKRN3) Overexpression in the Arcuate Nucleus of Postpubertal Female Mice on the HPG Axis

Context: Familial central precocious puberty (CPP) is a prevalent form (about 27.5%) of precocious puberty. Loss-of-function mutations in two maternally imprinted genes, MKRN3 and DLK1, were identified in families with CPP showing dominant autosomal inheritance with paternal transmission. Maternally transmitted CPP was previously demonstrated in up to 60% of families using pedigree analysis, however, no definitive genetic abnormality has been identified so far.
Objectives: To estimate the prevalence of familial cases in a multiethnic cohort with CPP. To characterize the genetic basis and the mode of inheritance of the affected families. To compare clinical and hormonal features of patients with familial CPP due to different modes of transmission.
Patients and Methods: Clinical and hormonal data were obtained from medical registries of 495 patients with CPP and no brain MRI alterations. Familial CPP was defined as the presence of one or more close relatives with CPP or precocious menarche (≤9 yr). Sanger sequencing of MKRN3 and DLK1 was performed in 427 index cases. Targeted gene panel sequencing was performed in 79 cases, while whole exome sequencing was performed in 101 cases from 36 families
Results: Among 495 index cases, 159 had familial CPP (31%). The mode of transmission of CPP was identified as paternal in 58 (35%), maternal in 59 (38%), indeterminate in 34 (22%), and both maternal and paternal transmission in 8 (5%). Most families with paternal or maternal transmission had 2 generations known to be affected (51 and 77%, respectively). Notably, 67% of index cases with maternally transmitted CPP had their mother affected. In girls with CPP, the median age of thelarche was 6.5 yr in the paternally transmitted group, 6.9 yr in the maternally transmitted group, and 7.3 yr in the indeterminate group (p= 0.547). Median bone age advancement was 2.1, 2.3 and 1.2 yr, respectively (p= 0.013). Basal LH levels were higher in girls with paternally transmitted CPP (p= 0.047). Among those with paternally transmitted CPP, MKRN3 and DLK1 mutations were identified in, respectively, 63.8% and 10.3% of the families.
Conclusions: A significant prevalence (31%) of familial CPP was demonstrated in a multiethnic cohort. Maternally transmitted CPP represented the most frequent form of familial CPP (38%). MKRN3 loss-of-function mutations were responsible for most paternally transmitted CPP cases (63.8%), followed by DLK1 loss-of-function mutations (10.3%).

Clinical, Hormonal and Genetic Characterization of Familial Central Precocious Puberty

Objectives: The 17β-hydroxysteroid dehydrogenase type 3 (17βHSD3) enzyme, expressed in the testes, converts androstenedione to testosterone. Deficiency of 17βHSD3 causes a 46,XY difference of sex development (DSD). Until recently, just 12 cases of 17βHSD3 deficiency had been reported in the literature in the United States. We report a series of 16 patients diagnosed at five children's hospitals in the United States.
Methods: We performed a multi-center chart review of patients with 17βHSD3 deficiency diagnosed based on hormone values and/or sequencing and deletion/duplication analysis of the HSD17B3 gene.
Results: Sixteen patients were identified, ranging in age at diagnosis from birth to 22.3 years (average age 9.5 years). One patient, assigned female at birth, was diagnosed due to a positive family history of the condition. Three patients underwent testing due to discordance between the sex predicted by prenatal cell-free DNA testing and the external genital appearance on prenatal ultrasound or at birth (two were assigned female, one was assigned male). An additional four patients had non-binary (atypical) appearing external genitalia at birth (two assigned female, two assigned male). Two children assigned female at birth were diagnosed after undescended testes were identified during hernia repair in early childhood. Six patients who were assigned female at birth came to medical attention peri-pubertally due to signs of excess/undesired androgen effects and/or primary amenorrhea. Data on gender identity is limited by the current age of many of the patients, but as of most recent follow-up, one patient assigned male and one assigned female are reported to be exploring their gender identities.
Four patients had a prior diagnosis of complete or partial androgen insensitivity syndrome and 8 had a prior diagnosis of 46,XY DSD of unknown etiology. Eleven patients had genetic testing confirming pathogenic or likely pathogenic variants in the HSD17B3 gene (two identified on whole exome, one on a DSD-specific multi-gene panel, and the remaining 8 on single gene testing). Of the patients without genetically confirmed 17βHSD3 deficiency, one had a clinical diagnosis due to a genetically-confirmed diagnosis in a sibling. The remaining four declined or have not completed genetic testing, but had hormonal testing consistent with the diagnosis.
Conclusions: 17βHSD3 deficiency is likely much more common in the US than previously appreciated, and can present at any age with a range of physical findings. Accurate diagnosis is important, as the broad category of 46,XY DSD encompasses a wide spectrum of gonadal malignancy risk, potential for pubertal hormone function and fertility, and gender identity outcomes. We suggest evaluating for this potential diagnosis with genetic and/or hormonal testing in cases of 46,XY DSD with absent uterus.

Case Series of 16 Patients With 17β-Hydroxysteroid Dehydrogenase Type 3 Deficiency at Five Children's Hospitals in the United States

Introduction: Classic congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) is a rare autosomal recessive disease characterized by deficiency of cortisol and oftentimes aldosterone, with elevated adrenocorticotropic hormone (ACTH) and steroid precursors that are then shunted toward excess androgen production. A phase 2 study of adults with classic 21OHD demonstrated that crinecerfont—an oral, non-steroidal, selective corticotropin-releasing factor type 1 (CRF₁) receptor antagonist—substantially reduced elevated hormone markers after 14 days of treatment [1]. The current study evaluated the effect of crinecerfont in adolescents with classic 21OHD, who may be especially challenging to manage for multiple reasons including the hormonal changes associated with puberty [2].
Methods: Eligible adolescents (14─17 years of age) with classic 21OHD received open-label crinecerfont (50 mg BID) for 14 days. ACTH, 17-hydroxyprogesterone (17OHP), androstenedione, and testosterone were assessed over 24 hours prior to (baseline) and after 14 days of crinecerfont treatment. Morning window values were defined as the average of samples collected at 0700h and 1000h, before the morning glucocorticoid dose. The participants' glucocorticoid and fludrocortisone regimens were maintained stable during crinecerfont treatment.
Results: In 8 participants (3 males, 5 females, ages 14-16 years), baseline median hormone concentrations (based on morning window) were as follows: plasma ACTH, 226.2 pg/mL; serum 17OHP, 7703.7 ng/dL; serum androstenedione, 367.9 ng/dL; and in females, serum testosterone, 63.5 ng/dL. After 14 days of crinecerfont treatment, median percent reductions from baseline were: ACTH, -57.1%; 17OHP, -69.5%; androstenedione, -58.3%; and in females, testosterone, ‑76.2%. A ≥50% reduction from baseline to Day 14 in ACTH, 17OHP, and androstenedione was observed in 62.5%, 75.0%, and 50.0% of participants, respectively. Furthermore, 60% (3/5) of females had ≥50% reduction from baseline in testosterone. Thirteen treatment-emergent adverse events (TEAEs) were reported; all but 1 (bruising) were assessed as mild. The majority of TEAEs were assessed as unrelated by the investigator, with the most common being headache (n=2). There were no serious AEs, discontinuations due to AEs, or safety concerns related to routine laboratory tests, vital signs, or electrocardiograms.
Conclusions: In adolescents with classic 21OHD, substantial median reductions (57-76%) in adrenal androgens and androgen precursors were observed after 14 days of crinecerfont treatment, consistent with reported results from a similar study of adults with classic 21OHD. Further studies are warranted to evaluate whether longer-term treatment with crinecerfont can allow for lower, more physiologic glucocorticoid dosing by directly reducing adrenal androgens, and thereby improving clinical outcomes (weight, metabolic risk, growth/development, fertility, etc.), via antagonism of the CRF₁ receptor.

Crinecerfont (NBI-74788), a Novel CRF1 Receptor Antagonist, Lowers Adrenal Androgens and Precursors in Adolescents with Classic Congenital Adrenal Hyperplasia

Objectives:
Vosoritide is a C-type natriuretic peptide analog which binds its receptor on chondrocytes, leading to increased chondrocyte proliferation and differentiation via its inhibition of the ERK1/2-MAPK pathway. It was recently approved for increasing linear growth in children with achondroplasia. Our study aims to assess the safety and efficacy of vosoritide in children with genetic mutations in 6 categories: hypochondroplasia, Rasopathies (including Noonan syndrome), aggrecan deficiency, carriers of heterozygous NPR2 mutations, CNP deficiency, and SHOX deficiency.
Methods:
This is a prospective, Phase II study with a goal N of 35 subjects. All subjects must have a documented genetic mutation in one of the 6 categories, be prepubertal between the ages of 3 and 11 for boys and 3 and 10 for girls, and have a height < -2.25 SD. Subjects are followed for a 6-month observation period to establish a baseline annualized growth velocity (AGV) and then receive daily subcutaneous vosoritide (15 mcg/kg/day) for 12 months. The primary outcomes are rate of AEs and change in AGV from baseline. Pharmacokinetic studies and pharmacodynamic studies, using cGMP production as a pharmacodynamic marker, are completed at Day 1 and Months 6 and 12.
Results:
To date, 27 subjects have enrolled in the trial (20 hypochondroplasia, 3 Noonan, 2 NPR2, 2 Aggrecan). Median baseline height is -3.1 SD (IQR -3.6, -2.6). 14 subjects have initiated on vosoritide, and 10 completed 6 months of therapy. Median increase in AGV was 3.9 cm/yr (IQR 2.2, 4.6). The two subjects with NPR2 mutations had increased AGVs of 4.4 and 9.2 cm/yr. The two subjects with Noonan syndrome had an increase in AGV of 7.5 and 3.3 cm/yr. One subject with Noonan syndrome, a 4-year-old male, had previously been treated with growth hormone (GH) for 2 years. His AGV was 6.7 cm/yr while on GH, 4.1 cm/yr during the 6-month observation period, and 11.6 cm/year during the first 6 months of vosoritide treatment. There were no serious adverse events related to vosoritide treatment. One subject had a brief episode of syncope with her first injection which self-resolved, and she continued on therapy with no further incidents. PK parameters were similar to previously published parameters for vosoritide in children with achondroplasia. In preliminary data, vosoritide Cmax and AUC correlated strongly with peak increase in cGMP (r=0.70, p=0.01;r=0.84, p<0.01, respectively) but not with increase in AGV (r=0.35, p=0.32).
Conclusions:
This is the first clinical trial of vosoritide for children with genetic short stature who do not have achondroplasia. Vosoritide treatment may work as a precision therapy to improve growth in multiple genetic conditions which interact with the ERK1/2-MAPK pathway.

A Prospective Clinical Trial of Vosoritide in Selected Genetic Causes of Short Stature

Background: Turner syndrome (TS) is a chromosomal anomaly occurring in females which presents with numerous medical and developmental manifestations including skeletal deformities. A specific developmental skeletal dyplasia seen in TS patients is Madelung deformity (MD), characterized by a growth deficiency of the distal radial physis that causes excessive radial inclination and volar tilt, with resultant ulnocarpal impaction. Surgical intervention, particularly in early stages, can halt progression of or reverse MD. Despite a considerable body of literature highlighting the presence of MD in TS, it is often underdiagnosed, in part due to a lack of data documenting the epidemiology and natural history. Objective: Analyze the frequency of diagnosis of MD in patients meeting key radiographic threshold criteria and document the time course of the deformity in these patients using these parameters. Methods: From a database of 264 patients with a confirmed diagnosis of TS, bone age radiographs for 187 (71%) were retrospectively measured against generally accepted cutoffs for MD using two parameters: ulnar tilt = UT and lunate subsidence = LS. For patients meeting general inclusion criteria of UT > 33° or LS < 0 mm, historical measurements of UT and LS were obtained using past bone ages and patients' charts were reviewed to determine if a clinical diagnosis of MD was made. Results: 7 patients (7/187 = 3.7%) met overall inclusion criteria and had radiographs consistent with Madelung Deformity. Of these 7, only one clinical diagnosis (14%) of MD was made; this patient underwent subsequent surgical intervention. The 6 undiagnosed patients had an average UT and LS of 40.5 degrees and -4.4 mm at the time of most recent bone age (~16.3 years old). The time-courses for 4 (UT) and 3 (LS) of the 6 non-surgical patients were plotted over time from 7.2 – 18.9 years of age. Patients crossed threshold at an average age of 15.3 and 13.6 years for UT and LS, respectively. Average maximum read-to-read growth rates of 5.3 degrees and 3.5 mm occurred at an average age of 15.3 (UT) and 14.2 years (LS). Conclusion: Our results suggest MD is significantly underdiagnosed in patients with TS, highlighting the need for better screening tools to make this diagnosis. This would include physical examination looking for the characteristic wrist deformity and careful analysis of bone age radiographs at an earlier age, when corrective surgery is more likely to succeed. We recommend careful scrutiny of yearly bone age radiographs from 10-15 years of age to ensure that the diagnosis of MD is not missed, and obtaining bilateral wrist radiographs (PA and Lateral views) for verification. Future research should focus on documenting disease progression using radiographic trends, as well as assessing how onset and progression are affected by therapeutic interventions such as growth hormone.

MKRN3 inhibits the reproductive axis by interacting with key hypothalamic substrates and targeting neurokinin B to degradation pathways.

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