How human genetic context can inform pathogenicity classification: FGFR1 variation in idiopathic hypogonadotropic hypogonadism.

Precision medicine requires precise genetic variant interpretation, yet many disease-associated genes have unresolved variants of unknown significance (VUS). We analyzed variants in a well-studied gene, FGFR1, a common cause of Idiopathic Hypogonadotropic Hypogonadism (IHH) and examined whether regional genetic enrichment of missense variants could improve variant classification. FGFR1 rare sequence variants (RSVs) were examined in a large cohort to (i) define regional genetic enrichment, (ii) determine pathogenicity based on the American College of Medical Genetics/Association for Molecular Pathology (ACMG/AMP) variant classification framework, and (iii) characterize the phenotype of FGFR1 variant carriers by variant classification. A total of 143 FGFR1 RSVs were identified in 175 IHH probands (n = 95 missense, n = 48 protein-truncating variants). FGFR1 missense RSVs showed regional enrichment across biologically well-defined domains: D1, D2, D3, and TK domains and linker regions (D2-D3, TM-TK). Using these defined regions of enrichment to augment the ACMG/AMP classification reclassifies 37% (20/54) of FGFR1 missense VUS as pathogenic or likely pathogenic (PLP). Non-proband carriers of FGFR1 missense VUS variants that were reclassified as PLP were more likely to express IHH or IHH-associated phenotypes [anosmia or delayed puberty] than non-proband carriers of FGFR1 missense variants that remained as VUS (76.9% vs 34.7%, p = 0.035). Using the largest cohort of FGFR1 variant carriers, we show that integration of regional genetic enrichment as moderate evidence for pathogenicity improves the classification of VUS and that reclassified variants correlated with phenotypic expressivity. The addition of regional genetic enrichment to the ACMG/AMP guidelines may improve clinical variant interpretation.

The p190 RhoGAPs, ARHGAP35, and ARHGAP5 are implicated in GnRH neuronal development: Evidence from patients with idiopathic hypogonadotropic hypogonadism, zebrafish, and in vitro GAP activity assay.

PURPOSE: The study aimed to identify novel genes for idiopathic hypogonadotropic hypogonadism (IHH). METHODS: A cohort of 1387 probands with IHH underwent exome sequencing and de novo, familial, and cohort-wide investigations. Functional studies were performed on 2 p190 Rho GTPase-activating proteins (p190 RhoGAP), ARHGAP35 and ARHGAP5, which involved in vivo modeling in larval zebrafish and an in vitro p190A-GAP activity assay. RESULTS: Rare protein-truncating variants (PTVs; n = 5) and missense variants in the RhoGAP domain (n = 7) in ARHGAP35 were identified in IHH cases (rare variant enrichment: PTV [unadjusted P = 3.1E-06] and missense [adjusted P = 4.9E-03] vs controls). Zebrafish modeling using gnrh3:egfp phenotype assessment showed that mutant larvae with deficient arhgap35a, the predominant ARHGAP35 paralog in the zebrafish brain, display decreased GnRH3-GFP+ neuronal area, a readout for IHH. In vitro GAP activity studies showed that 1 rare missense variant [ARHGAP35 p.(Arg1284Trp)] had decreased GAP activity. Rare PTVs (n = 2) also were discovered in ARHGAP5, a paralog of ARHGAP35; however, arhgap5 zebrafish mutants did not display significant GnRH3-GFP+ abnormalities. CONCLUSION: This study identified ARHGAP35 as a new autosomal dominant genetic driver for IHH and ARHGAP5 as a candidate gene for IHH. These observations suggest a novel role for the p190 RhoGAP proteins in GnRH neuronal development and integrity.

Burden Testing of Rare Variants Identified through Exome Sequencing via Publicly Available Control Data.

The genetic causes of many Mendelian disorders remain undefined. Factors such as lack of large multiplex families, locus heterogeneity, and incomplete penetrance hamper these efforts for many disorders. Previous work suggests that gene-based burden testing-where the aggregate burden of rare, protein-altering variants in each gene is compared between case and control subjects-might overcome some of these limitations. The increasing availability of large-scale public sequencing databases such as Genome Aggregation Database (gnomAD) can enable burden testing using these databases as controls, obviating the need for additional control sequencing for each study. However, there exist various challenges with using public databases as controls, including lack of individual-level data, differences in ancestry, and differences in sequencing platforms and data processing. To illustrate the approach of using public data as controls, we analyzed whole-exome sequencing data from 393 individuals with idiopathic hypogonadotropic hypogonadism (IHH), a rare disorder with significant locus heterogeneity and incomplete penetrance against control subjects from gnomAD (n = 123,136). We leveraged presumably benign synonymous variants to calibrate our approach. Through iterative analyses, we systematically addressed and overcame various sources of artifact that can arise when using public control data. In particular, we introduce an approach for highly adaptable variant quality filtering that leads to well-calibrated results. Our approach "re-discovered" genes previously implicated in IHH (FGFR1, TACR3, GNRHR). Furthermore, we identified a significant burden in TYRO3, a gene implicated in hypogonadotropic hypogonadism in mice. Finally, we developed a user-friendly software package TRAPD (Test Rare vAriants with Public Data) for performing gene-based burden testing against public databases.

Evaluating co-created patient-facing materials to increase understanding of genetic test results.

Patients often have difficulty understanding genetic test reports. Technical language and jargon can impede comprehension and limit patients using results to act on findings. One potential way to improve patient understanding of genetic test reports is to provide patient-facing materials. This study aimed to examine understandability and actionability of co-created patient-facing materials for genetic test results in a research context. We combined interprofessional perspectives and patient engagement to co-create patient-facing materials for patients undergoing research genetic testing for congenital hypogonadotropic hypogonadism (Kallmann syndrome). The iterative development process was guided by principles of health literacy and human-centered design (i.e., design thinking). Readability was assessed using eight validated algorithms. Patients and parents evaluated materials using a web-based survey. The gold standard Patient Education Materials Assessment Tool for print materials (PEMAT-P) was employed to measure understandability (content, style, use of numbers, organization, design, use of visual aids) and actionability. PEMAT-P scores >80% were considered high quality. Results were analyzed descriptively and correlations performed to identify relationships between education/health literacy and PEMAT-P ratings. A consensus score of eight algorithms indicated the materials were an 8th -9th grade reading level. Our findings are consistent with the U.S. Department of Health and Human Services 'average difficulty' classification (i.e., 7th-9th grade). In total, 61 patients/parents evaluated the materials. 'Visual Aids' received the lowest mean PEMAT-P rating (89%). All other parameters scored 90%-97%. PEMAT-P scores did not differ according to educational attainment (less than college vs. college or more, p = 0.28). Participants with adequate health literacy were more likely to approve of the 'organization' of information (p < 0.05). Respondents with low health literacy had more favorable views of 'visual aids' (p < 0.01). Involving patients in a co-creation process can produce high-quality patient-facing materials that are easier to understand.

Determinants of Power in Gene-Based Burden Testing for Monogenic Disorders.

Whole-exome sequencing has enabled new approaches for discovering genes associated with monogenic disorders. One such approach is gene-based burden testing, in which the aggregate frequency of "qualifying variants" is compared between case and control subjects for each gene. Despite substantial successes of this approach, the genetic causes for many monogenic disorders remain unknown or only partially known. It is possible that particular genetic architectures lower rates of discovery, but the influence of these factors on power has not been rigorously evaluated. Here, we leverage large-scale exome-sequencing data to create an empirically based simulation framework to evaluate the impact of key parameters (background variation rates, locus heterogeneity, mode of inheritance, penetrance) on power in gene-based burden tests in the context of monogenic disorders. Our results demonstrate that across genes, there is a wide range in sample sizes needed to achieve power due to differences in the background rate of rare variants in each gene. Increasing locus heterogeneity results in rapid increases in sample sizes needed to achieve adequate power, particularly when individual genes contribute to less than 5% of cases under a dominant model. Interestingly, incomplete penetrance as low as 10% had little effect on power due to the low prevalence of monogenic disorders. Our results suggest that moderate incomplete penetrance is not an obstacle in this gene-based burden testing approach but that dominant disorders with high locus heterogeneity will require large sample sizes. Our simulations also provide guidance on sample size needs and inform study design under various genetic architectures.

Phenotypic spectrum of POLR3B mutations: isolated hypogonadotropic hypogonadism without neurological or dental anomalies.

BACKGROUND: A constellation of neurodegenerative disorders exists (Gordon Holmes syndrome, 4H leucodystrophy, Boucher-Neuhauser syndrome) in which patients suffer from both neurological disease (typically manifested by ataxia) and reproductive failure (idiopathic hypogonadotropic hypogonadism (IHH)). POLR3B, which encodes the second largest subunit of RNA polymerase III (pol III), and POLR3A, which forms the pol III catalytic centre, are associated with 4H leucodystrophy. METHODS: Whole exome sequencing was performed on a large cohort of subjects with IHH (n=565). Detailed neuroendocrine studies were performed in some individuals within this cohort. RESULTS: Four individuals (two of them siblings) were identified with two rare nucleotide variants in POLR3B. On initial evaluation, all subjects were free of neurological disease. One patient underwent treatment with exogenous pulsatile gonadotropin-releasing hormone for 8 weeks which failed to result in normalisation of his sex steroid milieu due to pituitary resistance. CONCLUSIONS: These findings suggest that the spectrum of phenotypes resulting from POLR3B mutations is wider than previously believed and that POLR3B can be associated exclusively with disorders characterised by abnormal gonadotropin secretion.

Defective FGFR1 Signaling Disrupts Glucose Regulation: Evidence From Humans With FGFR1 Mutations.

Context: Activation of fibroblast growth factor receptor 1 (FGFR1) signaling improves the metabolic health of animals and humans, while inactivation leads to diabetes in mice. Direct human genetic evidence for the role of FGFR1 signaling in human metabolic health has not been fully established. Objective: We hypothesized that individuals with naturally occurring FGFR1 variants ("experiments of nature") will display glucose dysregulation. Methods: Participants with rare FGFR1 variants and noncarrier controls. Using a recall-by-genotype approach, we examined the ß-cell function and insulin sensitivity of 9 individuals with rare FGFR1 deleterious variants compared to 27 noncarrier controls, during a frequently sampled intravenous glucose tolerance test at the Reproductive Endocrine Unit and the Harvard Center for Reproductive Medicine, Massachusetts General Hospital. FGFR1-mutation carriers displayed higher ß-cell function in the face of lower insulin sensitivity compared to controls. Conclusion: These findings suggest that impaired FGFR1 signaling may contribute to an early insulin resistance phase of diabetes pathogenesis and support the candidacy of the FGFR1 signaling pathway as a therapeutic target for improving the human metabolic health.

Contributions of common genetic variants to constitutional delay of puberty and idiopathic hypogonadotropic hypogonadism.

CONTEXT: Constitutional delay of puberty (CDP) is highly heritable, but the genetic basis for CDP is largely unknown. Idiopathic hypogonadotropic hypogonadism (IHH) can be caused by rare genetic variants, but in about half of cases, no rare-variant cause is found. OBJECTIVE: To determine whether common genetic variants that influence pubertal timing contribute to CDP and IHH. DESIGN: Case-control study. PARTICIPANTS: 80 individuals with CDP; 301 with normosmic IHH, and 348 with Kallmann syndrome; control genotyping data from unrelated studies. MAIN OUTCOME MEASURES: Polygenic scores (PGS) based on genome-wide association studies for timing of male pubertal hallmarks and age at menarche (AAM). RESULTS: The CDP cohort had higher PGS for male pubertal hallmarks and for AAM compared to controls (for male hallmarks, Cohen's d = 0.85, p = 1 × 10-16; for AAM, d = 0.67, p = 1 × 10-10). The normosmic IHH cohort also had higher PGS for male hallmarks compared to controls, but the difference was smaller (male hallmarks d = 0.20, p = 0.003; AAM d = 0.10, p = 0.055). No differences were seen for the KS cohort compared to controls (male hallmarks d = 0.04, p = 0.45; AAM d = -0.03, p = 0.86). CONCLUSIONS: Common genetic variants that influence pubertal timing in the general population contribute strongly to the genetics of CDP, weakly to normosmic IHH, and potentially not at all to KS. These findings demonstrate that the common-variant genetics of CDP and normosmic IHH are largely but not entirely distinct.

Use of genetic models of idiopathic hypogonadotrophic hypogonadism in mice and men to understand the mechanisms of disease.

Mutations in the genes encoding the neuropeptides kisspeptin and neurokinin B, as well as their receptors, are associated with gonadotrophin-releasing hormone (GnRH) deficiency and a failure to initiate and/or progress through puberty. Although the total number of patients studied to date is small, mutations in the kisspeptin pathway appear to result in lifelong GnRH deficiency. Mice with mutations in kisspeptin and the kisspeptin receptor, Kiss1(-/-) and Kiss1r(-/-), respectively, appear to be phenocopies of the human with abnormal sexual maturation and infertility. In contrast, mutations in the neurokinin B pathway lead to a more variable adult reproductive phenotype, with a subset of hypogonadotrophic individuals demonstrating paradoxical recovery of reproductive function later in life. While 'reversal' remains poorly understood, the ability to recover reproductive function indicates that neurokinin B may play different roles in the initiation of sexual maturation compared with the maintenance of adult reproductive function. Mice with mutations in the gene encoding the neurokinin B receptor, Tacr3, have abnormal oestrous cycles and subfertility but, similar to their human counterparts, appear less severely affected than mice with kisspeptin deficiency. Further investigations into the interaction between the kisspeptin and neurokinin B pathways will reveal key insights into how GnRH neuronal modulation occurs at puberty and throughout reproductive life.

Prevalence and Phenotypic Effects of Copy Number Variants in Isolated Hypogonadotropic Hypogonadism.

CONTEXT: The genetic architecture of isolated hypogonadotropic hypogonadism (IHH) has not been completely defined. OBJECTIVE: To determine the role of copy number variants (CNVs) in IHH pathogenicity and define their phenotypic spectrum. METHODS: Exome sequencing (ES) data in IHH probands (n = 1394) (Kallmann syndrome [IHH with anosmia; KS], n = 706; normosmic IHH [nIHH], n = 688) and family members (n = 1092) at the Reproductive Endocrine Unit and the Center for Genomic Medicine of Massachusetts General Hospital were analyzed for CNVs and single nucleotide variants (SNVs)/indels in 62 known IHH genes. IHH subjects without SNVs/indels in known genes were considered "unsolved." Phenotypes associated with CNVs were evaluated through review of patient medical records. A total of 29 CNVs in 13 genes were detected (overall IHH cohort prevalence: ~2%). Almost all (28/29) CNVs occurred in unsolved IHH cases. While some genes (eg, ANOS1 and FGFR1) frequently harbor both CNVs and SNVs/indels, the mutational spectrum of others (eg, CHD7) was restricted to SNVs/indels. Syndromic phenotypes were seen in 83% and 63% of IHH subjects with multigenic and single gene CNVs, respectively. CONCLUSION: CNVs in known genes contribute to ~2% of IHH pathogenesis. Predictably, multigenic contiguous CNVs resulted in syndromic phenotypes. Syndromic phenotypes resulting from single gene CNVs validate pleiotropy of some IHH genes. Genome sequencing approaches are now needed to identify novel genes and/or other elusive variants (eg, noncoding/complex structural variants) that may explain the remaining missing etiology of IHH.

Kisspeptin Overcomes GnRH Neuronal Suppression Secondary to Hyperprolactinemia in Humans.

CONTEXT: Hyperprolactinemia suppresses gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) pulses. The hypothalamic neuropeptide kisspeptin potently stimulates the secretion of GnRH. The effects of exogenous kisspeptin administration on GnRH pulse generation in the setting of hyperprolactinemia have not previously been explored. OBJECTIVE: This work aimed to examine the effects of kisspeptin on GnRH secretion, as reflected by LH secretion, in women with hyperprolactinemia. METHODS: Women with hyperprolactinemia (n = 11) participated in two 12-hour visits. Before study visits, participants underwent washout of dopamine agonist and/or combined oral contraceptive. Frequent blood sampling was performed (1 sample was collected every 10 minutes). Visit 1 involved no intervention, to examine baseline LH pulsatility. During visit 2, kisspeptin 112-121 (0.24 nmol/kg) was administered every 1 hour, for 10 hours. At hour 11, one intravenous bolus of GnRH (75 ng/kg) was administered. RESULTS: Repetitive intravenous bolus kisspeptin administration increased the total number of LH pulses in the setting of hyperprolactinemia. The interpulse interval declined during the same time frames. LH pulse amplitude did not change, but the mean LH rose. In 6 participants with progesterone levels suggestive of an anovulatory state, mean LH and estradiol levels increased significantly at visit 2. In the entire cohort, follicle-stimulating hormone and prolactin levels did not change significantly across the 2 visits. A total of 73% of subjects exhibited an LH pulse within 30 minutes of first kisspeptin dose. CONCLUSION: Kisspeptin is capable of stimulating hypothalamic GnRH-induced LH pulses in the setting of hyperprolactinemia.

Using Kisspeptin to Predict Pubertal Outcomes for Youth With Pubertal Delay.

CONTEXT: The management of youth with delayed puberty is hampered by difficulty in predicting who will eventually progress through puberty and who will fail to attain adult reproductive endocrine function. The neuropeptide kisspeptin, which stimulates gonadotropin-releasing hormone (GnRH) release, can be used to probe the integrity of the reproductive endocrine axis. OBJECTIVE: We sought to determine whether responses to kisspeptin can predict outcomes for individuals with pubertal delay. DESIGN, SETTING, AND PARTICIPANTS: We conducted a longitudinal cohort study in an academic medical center of 16 children (3 girls and 13 boys) with delayed or stalled puberty. INTERVENTION AND OUTCOME MEASURES: Children who had undergone kisspeptin- and GnRH-stimulation tests were followed every 6 months for clinical evidence of progression through puberty. Inhibin B was measured in boys. A subset of participants underwent exome sequencing. RESULTS: All participants who had responded to kisspeptin with a rise in luteinizing hormone (LH) of 0.8 mIU/mL or greater subsequently progressed through puberty (n = 8). In contrast, all participants who had exhibited LH responses to kisspeptin ≤ 0.4 mIU/mL reached age 18 years without developing physical signs of puberty (n = 8). Thus, responses to kisspeptin accurately predicted later pubertal outcomes (P = .0002). Moreover, the kisspeptin-stimulation test outperformed GnRH-stimulated LH, inhibin B, and genetic testing in predicting pubertal outcomes. CONCLUSION: The kisspeptin-stimulation can assess future reproductive endocrine potential in prepubertal children and is a promising novel tool for predicting pubertal outcomes for children with delayed puberty.

Hypothalamic Reproductive Endocrine Pulse Generator Activity Independent of Neurokinin B and Dynorphin Signaling.

CONTEXT: Kisspeptin-neurokinin B (NKB)-dynorphin neurons are critical regulators of the hypothalamic-pituitary-gonadal axis. NKB and dynorphin are hypothesized to influence the frequency of GnRH pulses, whereas kisspeptin is hypothesized to be a generator of the GnRH pulse. How these neuropeptides interact remains unclear. OBJECTIVE: To probe the role of NKB in GnRH pulse generation and to determine the interactions between NKB, kisspeptin, and dynorphin in humans and mice with a complete absence of NKB. DESIGN: Case/control. SETTING: Academic medical center. PARTICIPANTS: Members of a consanguineous family bearing biallelic loss-of-function mutations in the gene encoding NKB and NKB-deficient mice. INTERVENTIONS: Frequent blood sampling to characterize neuroendocrine profile and administration of kisspeptin, GnRH, and naloxone, a nonspecific opioid receptor antagonist used to block dynorphin. MAIN OUTCOME MEASURES: LH pulse characteristics. RESULTS: Humans lacking NKB demonstrate slow LH pulse frequency, which can be increased by opioid antagonism. Mice lacking NKB also demonstrate impaired LH secretion, which can be augmented with an identical pharmacologic manipulation. Both mice and humans with NKB deficiency respond to exogenous kisspeptin. CONCLUSION: The preservation of LH pulses in the absence of NKB and dynorphin signaling suggests that both peptides are dispensable for GnRH pulse generation and kisspeptin responsiveness. However, NKB and dynorphin appear to have opposing roles in the modulation of GnRH pulse frequency.

Relation of endothelial function to cardiovascular risk in women with sedentary occupations and without known cardiovascular disease.

Our purpose was to determine predictors of endothelial function and potential association with cardiovascular risk in women with sedentary occupations, in whom obesity-associated risk factors may contribute to excess morbidity and mortality. Ninety consecutive women (age range 22 to 63 years, 22 overweight (body mass index [BMI] > or =25 to 29.9 kg/m(2)) and 42 obese (BMI > or = 30 kg/m(2)), had vital signs, lipids, insulin, glucose, high-sensitivity C-reactive protein, and sex hormones measured. Endothelial function was determined using brachial artery flow-mediated dilation after 5 minutes of forearm ischemia. Treadmill stress testing was performed with gas exchange analysis at peak exercise (peak oxygen consumption [Vo(2)]) to assess cardiorespiratory fitness. Brachial artery reactivity was negatively associated with Framingham risk score (r = -0.3542, p = 0.0007). Univariate predictors of endothelial function included peak Vo(2) (r = 0.4483, p <0.0001), age (r = -0.3420, p = 0.0010), BMI (r = -0.3065, p = 0.0035), and high-sensitivity C-reactive protein (r = -0.2220, p = 0.0400). Using multiple linear regression analysis with stepwise modeling, peak Vo(2) (p = 0.0003) was the best independent predictor of brachial artery reactivity, with age as the only other variable reaching statistical significance (p = 0.0436) in this model. In conclusion, endothelial function was significantly associated with cardiovascular risk in women with sedentary occupations, who were commonly overweight or obese. Even in the absence of routine exercise, cardiorespiratory fitness, rather than conventional risk factors or body mass, is the dominant predictor of endothelial function and suggests a modifiable approach to risk.

Predictors of endothelial function in employees with sedentary occupations in a worksite exercise program.

A sedentary workforce may be at increased risk for future cardiovascular disease. Exercise at the work site has been advocated, but effects on endothelium as a biomarker of risk and relation to weight loss, lipid changes, or circulating endothelial progenitor cells (EPCs) have not been reported. Seventy-two office and laboratory employees (58 women; average age 45 years, range 22 to 62; 26 with body mass index values >30 kg/m(2)) completed 3 months of participation in the National Heart, Lung, and Blood Institute's Keep the Beat program, with the determination of vital signs, laboratory data, and peak oxygen consumption (VO(2)) during treadmill exercise. Brachial artery endothelium was tested by flow-mediated dilation (FMD), which at baseline was inversely associated with Framingham risk score (r = -0.3689, p <0.0001). EPCs were quantified by colony assay. With exercise averaging 98 +/- 47 minutes each workweek, there was improvement in FMD (from 7.8 +/- 3.4% to 8.5 +/- 3.0%, p = 0.0096) and peak VO(2) (+1.2 +/- 3.1 ml O(2)/kg/min, p = 0.0028), with reductions in diastolic blood pressure (-2 +/- 8 mm Hg, p = 0.0478), total cholesterol (-8 +/- 25 mg/dl, p = 0.0131), and low-density lipoprotein cholesterol (-7 +/- 19 mg/dl, p = 0.0044) but with a marginal reduction in weight (-0.5 +/- 2.1 kg, p = 0.0565). By multiple regression modeling, lower baseline FMD, greater age, reductions in total and low-density lipoprotein cholesterol and diastolic blood pressure, and increases in EPC colonies and peak VO(2) were jointly statistically significant predictors of change in FMD and accounted for 47% of the variability in FMD improvement with program participation. Results were similar when modeling was performed for women only. In contrast, neither adiposity at baseline nor change in weight was a predictor of improved endothelial function. In conclusion, daily exercise achievable at their work sites by employees with sedentary occupations improves endothelial function, even with the absence of weight loss, which may decrease cardiovascular risk, if sustained.

Assessing Sex Steroid Influence on Kisspeptin Responsiveness in Idiopathic Hypogonadotropic Hypogonadism.

CONTEXT: Individuals with idiopathic hypogonadotropic hypogonadism (IHH), even those with evidence of some hypothalamic reproductive endocrine activity, fail to complete puberty and fail to respond to physiologic doses of kisspeptin. OBJECTIVE: This case series examined whether treatment with sex steroids could stimulate kisspeptin responsiveness in patients with IHH. DESIGN: This was a case series. SETTING: This study was conducted at an academic medical center. PARTICIPANTS: Seven patients with IHH were studied. INTERVENTIONS: Participants, both on and off sex steroid therapy, underwent frequent blood sampling to measure LH at baseline, in response to kisspeptin and GnRH. MAIN OUTCOME MEASURES: The main outcome measure was LH responses to kisspeptin on and off sex steroids. RESULTS: All participants responded to exogenous GnRH, but no participant responded to exogenous kisspeptin. Sex steroid treatment did not modify responsiveness to kisspeptin. CONCLUSIONS: The functional impairment of the GnRH neuronal network in patients with IHH, as evidenced by their inability to respond to a physiologic dose of kisspeptin, is observed in both sex steroid- deficient and sex steroid-replete states. In this case series, a normalized sex steroid milieu does not appear capable of overcoming the kisspeptin resistance of these patients.

Divergent responses to kisspeptin in children with delayed puberty.

BACKGROUND: The neuropeptide kisspeptin stimulates luteinizing hormone (LH) secretion in healthy adults but not in adults with idiopathic hypogonadotropic hypogonadism. We hypothesized that, in children presenting with delayed or stalled puberty, kisspeptin would elicit LH secretion in those children found on detailed nighttime neuroendocrine profiling to have evidence of emerging reproductive endocrine function. METHODS: Eleven boys and four girls were admitted overnight to assess LH secretion at baseline, after a single intravenous bolus of kisspeptin, and after a single intravenous bolus of gonadotropin-releasing hormone (GnRH). Subjects then received exogenous pulsatile GnRH for 6 days and returned for a second visit to measure responses to kisspeptin and GnRH after this pituitary "priming." Responses to kisspeptin and GnRH were also measured in 5 healthy men. RESULTS: Of the 15 children with delayed/stalled puberty, 6 exhibited at least one spontaneous LH pulse overnight; all of these subjects had clear responses to kisspeptin, as did one additional subject. Seven subjects had no response to kisspeptin, and one subject exhibited an intermediate response. In the children who responded to kisspeptin, the responses had features comparable to those of adult men. CONCLUSION: In this first report of kisspeptin administration to pediatric subjects to our knowledge, children with delayed/stalled puberty showed a wide range of responses, with some showing a robust response and others showing little to no response. Further follow-up will determine whether responses to kisspeptin predict future pubertal entry for children with delayed puberty. TRIAL REGISTRATION: ClinicalTrials.gov NCT01438034 and NCT01952782. FUNDING: NIH Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01 HD043341, R01 HD090071, P50 HD028138), NIH National Center for Advancing Translational (UL1 TR001102), NIH National Institute of Diabetes and Digestive and Kidney Diseases (T32 DK007028), the Massachusetts General Hospital Executive Committee on Research Fund for Medical Discovery, Harvard Catalyst, Doris Duke Charitable Foundation (award 2013110), Charles H. Hood Foundation, Robert and Laura Reynolds MGH Research Scholar Program, and Harvard University. These funding sources had no role in the design of this study and did not have any role in conducting the study, analyses, interpretation of the data, or the decision to submit results.

Continuous Kisspeptin Administration in Postmenopausal Women: Impact of Estradiol on Luteinizing Hormone Secretion.

Context: Kisspeptin stimulates the reproductive endocrine cascade in both men and women. Circulating sex steroids are thought to modulate the ability of kisspeptin to stimulate gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) release. Objective: To probe the effects of sex steroids on kisspeptin-stimulated GnRH-induced LH pulses. Participants: Eight healthy postmenopausal women. Intervention: Subjects underwent every-10-minute blood sampling to measure GnRH-induced LH secretion at baseline and in response to a continuous kisspeptin infusion (12.5 µg/kg/h) over 24 hours. A subset of the participants also received kisspeptin (0.313 µg/kg) and GnRH (75 ng/kg) intravenous boluses. Results: Postmenopausal women are resistant to the stimulatory effect of continuous kisspeptin on LH secretion. Postmenopausal women receiving estradiol replacement therapy are also resistant to kisspeptin initially, but they demonstrate a significant increase in LH pulse amplitude in direct proportion to the circulating estradiol concentration and duration of kisspeptin administration. Conclusions: Kisspeptin administration has complex effects on GnRH, and by extension, on LH secretion. The ability of kisspeptin to affect LH secretion can be modulated by the ambient sex-steroid milieu in a time- and dose-dependent manner.

SMCHD1 mutations associated with a rare muscular dystrophy can also cause isolated arhinia and Bosma arhinia microphthalmia syndrome.

Arhinia, or absence of the nose, is a rare malformation of unknown etiology that is often accompanied by ocular and reproductive defects. Sequencing of 40 people with arhinia revealed that 84% of probands harbor a missense mutation localized to a constrained region of SMCHD1 encompassing the ATPase domain. SMCHD1 mutations cause facioscapulohumeral muscular dystrophy type 2 (FSHD2) via a trans-acting loss-of-function epigenetic mechanism. We discovered shared mutations and comparable DNA hypomethylation patterning between these distinct disorders. CRISPR/Cas9-mediated alteration of smchd1 in zebrafish yielded arhinia-relevant phenotypes. Transcriptome and protein analyses in arhinia probands and controls showed no differences in SMCHD1 mRNA or protein abundance but revealed regulatory changes in genes and pathways associated with craniofacial patterning. Mutations in SMCHD1 thus contribute to distinct phenotypic spectra, from craniofacial malformation and reproductive disorders to muscular dystrophy, which we speculate to be consistent with oligogenic mechanisms resulting in pleiotropic outcomes.

Kisspeptin Responsiveness Signals Emergence of Reproductive Endocrine Activity: Implications for Human Puberty.

CONTEXT: Some patients with idiopathic hypogonadotropic hypogonadism (IHH) undergo spontaneous activation of their hypothalamic-pituitary-gonadal axis resulting in normalization of steroidogenesis and/or gametogenesis, a phenomenon termed reversal. OBJECTIVE: To assess the responsiveness of the GnRH neuronal network to exogenous kisspeptin administration in IHH patients who have undergone reversal. PARTICIPANTS: Six men with congenital IHH and evidence for reversal. INTERVENTION: Subjects underwent q10 min blood sampling to measure GnRH-induced LH secretion at baseline and in response to iv boluses of kisspeptin (0.24-2.4 nmol/kg) and GnRH (75 ng/kg). RESULTS: Individuals with sustained reversal of their hypogonadotropism (spontaneous LH pulses) responded to exogenous kisspeptin with a GnRH-induced LH pulse. Individuals who had reversal but then subsequently suffered relapse of their IHH (loss of spontaneous LH pulsatility) did not respond to kisspeptin. CONCLUSIONS: The ability of kisspeptin to stimulate a GnRH-induced LH pulse correlates with the presence of endogenous LH pulses. These data suggest that reversal of hypogonadotropism, and by extension sexual maturation, may be due to the acquisition of kisspeptin responsiveness.