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.

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.

Prevalence of Deleterious Variants in MC3R in Patients With Constitutional Delay of Growth and Puberty.

CONTEXT: The melanocortin 3 receptor (MC3R) has recently emerged as a critical regulator of pubertal timing, linear growth, and the acquisition of lean mass in humans and mice. In population-based studies, heterozygous carriers of deleterious variants in MC3R report a later onset of puberty than noncarriers. However, the frequency of such variants in patients who present with clinical disorders of pubertal development is currently unknown. OBJECTIVE: This work aimed to determine whether deleterious MC3R variants are more frequently found in patients clinically presenting with constitutional delay of growth and puberty (CDGP) or normosmic idiopathic hypogonadotropic hypogonadism (nIHH). METHODS: We examined the sequence of MC3R in 362 adolescents with a clinical diagnosis of CDGP and 657 patients with nIHH, experimentally characterized the signaling properties of all nonsynonymous variants found and compared their frequency to that in 5774 controls from a population-based cohort. Additionally, we established the relative frequency of predicted deleterious variants in individuals with self-reported delayed vs normally timed menarche/voice-breaking in the UK Biobank cohort. RESULTS: MC3R loss-of-function variants were infrequent but overrepresented in patients with CDGP (8/362 [2.2%]; OR = 4.17; P = .001). There was no strong evidence of overrepresentation in patients with nIHH (4/657 [0.6%]; OR = 1.15; P = .779). In 246 328 women from the UK Biobank, predicted deleterious variants were more frequently found in those self-reporting delayed (aged ≥16 years) vs normal age at menarche (OR = 1.66; P = 3.90E-07). CONCLUSION: We have found evidence that functionally damaging variants in MC3R are overrepresented in individuals with CDGP but are not a common cause of this phenotype.

Heterozygous mutations in SOX2 may cause idiopathic hypogonadotropic hypogonadism via dominant-negative mechanisms.

Pathogenic SRY-box transcription factor 2 (SOX2) variants typically cause severe ocular defects within a SOX2 disorder spectrum that includes hypogonadotropic hypogonadism. We examined exome-sequencing data from a large, well-phenotyped cohort of patients with idiopathic hypogonadotropic hypogonadism (IHH) for pathogenic SOX2 variants to investigate the underlying pathogenic SOX2 spectrum and its associated phenotypes. We identified 8 IHH individuals harboring heterozygous pathogenic SOX2 variants with variable ocular phenotypes. These variant proteins were tested in vitro to determine whether a causal relationship between IHH and SOX2 exists. We found that Sox2 was highly expressed in the hypothalamus of adult mice and colocalized with kisspeptin 1 (KISS1) expression in the anteroventral periventricular nucleus of adult female mice. In vitro, shRNA suppression of mouse SOX2 protein in Kiss-expressing cell lines increased the levels of human kisspeptin luciferase (hKiss-luc) transcription, while SOX2 overexpression repressed hKiss-luc transcription. Further, 4 of the identified SOX2 variants prevented this SOX2-mediated repression of hKiss-luc. Together, these data suggest that pathogenic SOX2 variants contribute to both anosmic and normosmic forms of IHH, attesting to hypothalamic defects in the SOX2 disorder spectrum. Our study describes potentially novel mechanisms contributing to SOX2-related disease and highlights the necessity of SOX2 screening in IHH genetic evaluation irrespective of associated ocular defects.

A functional spectrum of PROKR2 mutations identified in isolated hypogonadotropic hypogonadism.

Isolated hypogonadotropic hypogonadism (IHH) is a rare disease with hypogonadism and infertility caused by the defects in embryonic migration of hypothalamic gonadotropin-releasing hormone (GnRH) neurons, hypothalamic GnRH secretion or GnRH signal transduction. PROKR2 gene, encoding a G-protein coupled receptor PROKR2, is one of the most frequently mutated genes identified in IHH patients. However, the functional consequences of several PROKR2 mutants remain elusive. In this study, we systematically analyzed the Gαq, Gαs and ERK1/2 signaling of 23 IHH-associated PROKR2 mutations which are yet to be functionally characterized. We demonstrate that blockage of Gαq, instead of MAPK/ERK pathway, inhibited PROK2-induced migration of PROKR2-expressing cells, implying that PROKR2-related IHH results primarily due to Gαq signaling pathway disruption. Combined with previous reports, we categorized a total of 63 IHH-associated PROKR2 mutations into four distinct groups according Gαq pathway functionality: (i) neutral (N, >80% activity); (ii) low pathogenicity (L, 50-80% activity); (iii) medium pathogenicity (M, 20-50% activity) and (iv) high pathogenicity (H, <20% activity). We further compared the cell-based functional results with in silico mutational prediction programs. Our results indicated that while Sorting Intolerant from Tolerant predictions were accurate for transmembrane region mutations, mutations localized in the intracellular and extracellular domains were accurately predicted by the Combined Annotation Dependent Depletion prediction tool. Our results thus provide a functional database that can be used to guide diagnosis and appropriate genetic counseling in IHH patients with PROKR2 mutations.

Reproductive Phenotypes and Genotypes in Men With IHH.

CONTEXT: Isolated hypogonadotropic hypogonadism (IHH) is phenotypically and genetically heterogeneous. OBJECTIVE: This work aimed to determine the correlation between genotypic severity with pubertal and neuroendocrine phenotypes in IHH men. METHODS: A retrospective study was conducted (1980-2020) examining olfaction (Kallmann syndrome [KS] vs normosmic IHH [nHH]), baseline testicular volume (absent vs partial puberty), neuroendocrine profiling (pulsatile vs apulsatile luteinizing hormone [LH] secretion), and genetic variants in 62 IHH-associated genes through exome sequencing (ES). RESULTS: In total, 242 men (KS: n = 131 [54%], nHH: n = 111 [46%]) were included. Men with absent puberty had significantly lower gonadotropin levels (P < .001) and were more likely to have undetectable LH (P < .001). Logistic regression showed partial puberty as a statistically significant predictor of pulsatile LH secretion (R2 = 0.71, P < .001, OR: 10.8; 95% CI, 3.6-38.6). Serum LH of 2.10 IU/L had a 95% true positive rate for predicting LH pulsatility. Genetic analyses in 204 of 242 IHH men with ES data available revealed 36 of 204 (18%) men carried protein-truncating variants (PTVs) in 12 IHH genes. Men with absent puberty and apulsatile LH were enriched for oligogenic PTVs (P < .001), with variants in ANOS1 being the predominant PTV in this genotype-phenotype association. Men with absent puberty were enriched for ANOS1 PTVs compared to partial puberty counterparts (P = .002). PTVs in other IHH genes imparted more variable reproductive phenotypic severity. CONCLUSION: Partial puberty and LH greater than or equal to 2.10 IU/L are proxies for pulsatile LH secretion. ANOS1 PTVs confer severe reproductive phenotypes. Variable phenotypic severity in the face of severe genetic variants in other IHH genes point to significant neuroendocrine plasticity of the HPG axis in IHH men.

Reversible hypogonadotropic hypogonadism in men with the fertile eunuch/Pasqualini syndrome: A single-center natural history study.

Congenital hypogonadotropic hypogonadism (HH) is a heterogeneous genetic disorder characterized by disrupted puberty and infertility. In most cases, HH is abiding yet 10-15% undergo reversal. Men with HH and absent and partial puberty (i.e., testicular volume <4mL and >4mL respectively) have been well-studied, but the rare fertile eunuch (FE) variant remains poorly characterized. This natural history study of 240 men with HH delineates the clinical presentation, neuroendocrine profile, rate of reversal and genetics of the FE variant. We compared three HH groups: FE (n=38), absent puberty (n=139), and partial puberty (n=63). The FE group had no history of micropenis and 2/38 (5%) had cryptorchidism (p<0.0001 vs. other groups). The FE group exhibited higher rates of detectable gonadotropins, higher mean LH/FSH levels, and higher serum inhibin B levels (all p<0.0001). Neuroendocrine profiling showed pulsatile LH secretion in 30/38 (79%) of FE men (p<0.0001) and 16/36 (44%) FE men underwent spontaneous reversal of HH (p<0.001). The FE group was enriched for protein-truncating variants (PTVs) in GNRHR and FGFR1 and 4/30 (13%) exhibited oligogenic PTVs. Findings suggest men with the FE variant exhibit the mildest neuroendocrine defects of HH men and the FE sub-type represents the first identified phenotypic predictor for reversible HH.

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.

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.

Phenotypic continuum between Waardenburg syndrome and idiopathic hypogonadotropic hypogonadism in humans with SOX10 variants.

PURPOSE: SOX10 variants previously implicated in Waardenburg syndrome (WS) have now been linked to Kallmann syndrome (KS), the anosmic form of idiopathic hypogonadotropic hypogonadism (IHH). We investigated whether SOX10-associated WS and IHH represent elements of a phenotypic continuum within a unifying disorder or if they represent phenotypically distinct allelic disorders. METHODS: Exome sequencing from 1,309 IHH subjects (KS: 632; normosmic idiopathic hypogonadotropic hypogonadism [nIIHH]: 677) were reviewed for SOX10 rare sequence variants (RSVs). The genotypic and phenotypic spectrum of SOX10-related IHH (this study and literature) and SOX10-related WS cases (literature) were reviewed and compared with SOX10-RSV spectrum in gnomAD population. RESULTS: Thirty-seven SOX10-associated IHH cases were identified as follows: current study: 16 KS; 4 nIHH; literature: 16 KS; 1 nIHH. Twenty-three IHH cases (62%; all KS), had ≥1 known WS-associated feature(s). Moreover, five previously reported SOX10-associated WS cases showed IHH-related features. Four SOX10 missense RSVs showed allelic overlap between IHH-ascertained and WS-ascertained cases. The SOX10-HMG domain showed an enrichment of RSVs in disease states versus gnomAD. CONCLUSION: SOX10 variants contribute to both anosmic (KS) and normosmic (nIHH) forms of IHH. IHH and WS represent SOX10-associated developmental defects that lie along a unifying phenotypic continuum. The SOX10-HMG domain is critical for the pathogenesis of SOX10-related human disorders.

Increased Burden of Rare Sequence Variants in GnRH-Associated Genes in Women With Hypothalamic Amenorrhea.

CONTEXT: Functional hypothalamic amenorrhea (HA) is a common, acquired form of hypogonadotropic hypogonadism that occurs in the setting of energy deficits and/or stress. Variability in individual susceptibility to these stressors, HA heritability, and previous identification of several rare sequence variants (RSVs) in genes associated with the rare disorder, isolated hypogonadotropic hypogonadism (IHH), in individuals with HA suggest a possible genetic contribution to HA susceptibility. OBJECTIVE: We sought to determine whether the burden of RSVs in IHH-related genes is greater in women with HA than controls. DESIGN: We compared patients with HA to control women. SETTING: The study was conducted at secondary referral centers. PATIENTS AND OTHER PARTICIPANTS: Women with HA (n = 106) and control women (ClinSeq study; n = 468). INTERVENTIONS: We performed exome sequencing in all patients and controls. MAIN OUTCOME MEASURE(S): The frequency of RSVs in 53 IHH-associated genes was determined using rare variant burden and association tests. RESULTS: RSVs were overrepresented in women with HA compared with controls (P = .007). Seventy-eight heterozygous RSVs in 33 genes were identified in 58 women with HA (36.8% of alleles) compared to 255 RSVs in 41 genes among 200 control women (27.2%). CONCLUSIONS: Women with HA are enriched for RSVs in genes that cause IHH, suggesting that variation in genes associated with gonadotropin-releasing hormone neuronal ontogeny and function may be a major determinant of individual susceptibility to developing HA in the face of diet, exercise, and/or stress.

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.

TCF12 haploinsufficiency causes autosomal dominant Kallmann syndrome and reveals network-level interactions between causal loci.

Dysfunction of the gonadotropin-releasing hormone (GnRH) axis causes a range of reproductive phenotypes resulting from defects in the specification, migration and/or function of GnRH neurons. To identify additional molecular components of this system, we initiated a systematic genetic interrogation of families with isolated GnRH deficiency (IGD). Here, we report 13 families (12 autosomal dominant and one autosomal recessive) with an anosmic form of IGD (Kallmann syndrome) with loss-of-function mutations in TCF12, a locus also known to cause syndromic and non-syndromic craniosynostosis. We show that loss of tcf12 in zebrafish larvae perturbs GnRH neuronal patterning with concomitant attenuation of the orthologous expression of tcf3a/b, encoding a binding partner of TCF12, and stub1, a gene that is both mutated in other syndromic forms of IGD and maps to a TCF12 affinity network. Finally, we report that restored STUB1 mRNA rescues loss of tcf12 in vivo. Our data extend the mutational landscape of IGD, highlight the genetic links between craniofacial patterning and GnRH dysfunction and begin to assemble the functional network that regulates the development of the GnRH axis.

Hypogonadotropic hypogonadism due to variants in RAB3GAP2: expanding the phenotypic and genotypic spectrum of Martsolf syndrome.

Biallelic pathogenic variants in RAB3GAP2 cause Warburg Micro syndrome (WARBM) and Martsolf syndrome (MS), two rare, phenotypically overlapping disorders characterized by congenital cataracts, intellectual disability, and hypogonadism. Although the initial report documented hypergonadotropic hypogonadism (implying a gonadal defect), an adolescent girl with WARBM/MS was subsequently reported to have hypogonadotropic hypogonadism (implying a central defect in either the hypothalamus or anterior pituitary). However, in adult MS, hypogonadotropism has not been convincingly demonstrated. Additionally, the correlation between the pathogenic severity of variants in RAB3GAP2 and the phenotypic severity also remains unclear. Here we present a clinical report of a woman with congenital cataracts, apparent intellectual disability, and pubertal failure who underwent exome sequencing (ES) to determine a precise molecular diagnosis. Reproductive phenotypes reported previously in individuals with MS and the genotypic spectrum of previous RAB3GAP2 variants were also reviewed. The ES identified pathogenic compound heterozygous RAB3GAP2 variants (c.387-2A > G; p.(Arg428Glu)) combined with her phenotypic features, which enabled a unifying molecular diagnosis of MS. Reproductive evaluation confirmed a normosmic idiopathic hypogonadotropic hypogonadism. Review of the RAB3GAP2 allelic spectrum in WARBM/MS suggests that although variants resulting in complete abrogation of RAB3GAP2 protein function cause severe WARBM, variants associated with partially preserved RAB3GAP2 function cause milder MS. This report expands the genotypic and phenotypic spectrum of MS and demonstrates hypogonadotropic hypogonadism as a key pathophysiologic abnormality in MS. Genotype-phenotype associations of previously reported RAB3GAP2 variants indicate that variants that fully abolish RAB3GAP2 function result in WARBM, whereas MS is associated with variants of lesser severity with residual RAB3GAP2 function.

Insight Into the Ontogeny of GnRH Neurons From Patients Born Without a Nose.

CONTEXT: The reproductive axis is controlled by a network of gonadotropin-releasing hormone (GnRH) neurons born in the primitive nose that migrate to the hypothalamus alongside axons of the olfactory system. The observation that congenital anosmia (inability to smell) is often associated with GnRH deficiency in humans led to the prevailing view that GnRH neurons depend on olfactory structures to reach the brain, but this hypothesis has not been confirmed. OBJECTIVE: The objective of this work is to determine the potential for normal reproductive function in the setting of completely absent internal and external olfactory structures. METHODS: We conducted comprehensive phenotyping studies in 11 patients with congenital arhinia. These studies were augmented by review of medical records and study questionnaires in another 40 international patients. RESULTS: All male patients demonstrated clinical and/or biochemical signs of GnRH deficiency, and the 5 men studied in person had no luteinizing hormone (LH) pulses, suggesting absent GnRH activity. The 6 women studied in person also had apulsatile LH profiles, yet 3 had spontaneous breast development and 2 women (studied from afar) had normal breast development and menstrual cycles, suggesting a fully intact reproductive axis. Administration of pulsatile GnRH to 2 GnRH-deficient patients revealed normal pituitary responsiveness but gonadal failure in the male patient. CONCLUSIONS: Patients with arhinia teach us that the GnRH neuron, a key gatekeeper of the reproductive axis, is associated with but may not depend on olfactory structures for normal migration and function, and more broadly, illustrate the power of extreme human phenotypes in answering fundamental questions about human embryology.

A Balanced Translocation in Kallmann Syndrome Implicates a Long Noncoding RNA, RMST, as a GnRH Neuronal Regulator.

CONTEXT: Kallmann syndrome (KS) is a rare, genetically heterogeneous Mendelian disorder. Structural defects in KS patients have helped define the genetic architecture of gonadotropin-releasing hormone (GnRH) neuronal development in this condition. OBJECTIVE: Examine the functional role a novel structural defect affecting a long noncoding RNA (lncRNA), RMST, found in a KS patient. DESIGN: Whole genome sequencing, induced pluripotent stem cells and derived neural crest cells (NCC) from the KS patient were contrasted with controls. SETTING: The Harvard Reproductive Sciences Center, Massachusetts General Hospital Center for Genomic Medicine, and Singapore Genome Institute. PATIENT: A KS patient with a unique translocation, t(7;12)(q22;q24). INTERVENTIONS/MAIN OUTCOME MEASURE/RESULTS: A novel translocation was detected affecting the lncRNA, RMST, on chromosome 12 in the absence of any other KS mutations. Compared with controls, the patient's induced pluripotent stem cells and NCC provided functional information regarding RMST. Whereas RMST expression increased during NCC differentiation in controls, it was substantially reduced in the KS patient's NCC coincident with abrogated NCC morphological development and abnormal expression of several "downstream" genes essential for GnRH ontogeny (SOX2, PAX3, CHD7, TUBB3, and MKRN3). Additionally, an intronic single nucleotide polymorphism in RMST was significantly implicated in a genome-wide association study associated with age of menarche. CONCLUSIONS: A novel deletion in RMST implicates the loss of function of a lncRNA as a unique cause of KS and suggests it plays a critical role in the ontogeny of GnRH neurons and puberty.

Functional Hypogonadotropic Hypogonadism in Men: Underlying Neuroendocrine Mechanisms and Natural History.

CONTEXT: After completion of puberty a subset of men experience functional hypogonadotropic hypogonadism (FHH) secondary to excessive exercise or weight loss. This phenomenon is akin to hypothalamic amenorrhea (HA) in women, yet little is known about FHH in men. OBJECTIVE: To investigate the neuroendocrine mechanisms, genetics, and natural history underlying FHH. DESIGN: Retrospective study in an academic medical center. PARTICIPANTS: Healthy postpubertal men presenting with symptoms of hypogonadism in the setting of excessive exercise (>10 hours/week) or weight loss (>10% of body weight). Healthy age-matched men served as controls. INTERVENTIONS: Clinical assessment, biochemical and neuroendocrine profiling, body composition, semen analysis, and genetic evaluation of genes known to cause isolated GnRH deficiency. MAIN OUTCOME MEASURES: Reproductive hormone levels, endogenous GnRH-induced LH pulse patterns, and rare genetic variants. RESULTS: Ten men with FHH were compared with 18 age-matched controls. Patients had significantly lower body mass index, testosterone, LH, and mean LH pulse amplitudes yet normal LH pulse frequency, serum FSH, and sperm counts. Some patients exhibited nocturnal, sleep-entrained LH pulses characteristic of early puberty, and one FHH subject showed a completely apulsatile LH secretion. After decreased exercise and weight gain, five men with men had normalized serum testosterone levels, and symptoms resolved. Rare missense variants in NSMF (n = 1) and CHD7 (n = 1) were identified in two men with FHH. CONCLUSIONS: FHH is a rare, reversible form of male GnRH deficiency. LH pulse patterns in male FHH are similar to those observed in women with HA. This study expands the spectrum of GnRH deficiency disorders in men.

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.

Discordance in the Dependence on Kisspeptin Signaling in Mini Puberty vs Adolescent Puberty: Human Genetic Evidence.

Context: Hypothalamic kisspeptin signaling plays a critical role in the initiation and maintenance of reproductive function. Biallelic mutations in the coding sequence of KISS1R (GPR54) have been identified in patients with idiopathic hypogonadotropic hypogonadism, but it is unknown whether biallelic variants can also be associated with related reproductive disorders. Case Description: A missense homozygous variant (c.890G>T p.R297L) in KISS1R was identified in a child who presented with microphallus and bilateral cryptorchidism. This variant has been reported to reduce, but not abolish, postreceptor signaling in vitro. Biochemical evaluation during the neonatal period revealed low testosterone levels. By 11 years and 8 months, the boy began demonstrating increases in testicular volume. By 17 years and 3 months, his testicular volume was 20 mL; his penile length was 7.3 cm; and he had adult levels of circulating gonadotropins and testosterone. Conclusion: This case report associates biallelic loss-of-function mutations in KISS1R with normal timing of adolescent puberty. Because these coding sequence variants occurred in a patient with microphallus and cryptorchidism, they demonstrate different levels of dependence of the hypothalamic-pituitary-gonadal cascade on kisspeptin signaling at distinct times in the reproductive life span. The suppression of the hypothalamic-pituitary-gonadal cascade during early life but not adolescence suggests that the mini puberty of infancy depends more on kisspeptin-induced, gonadotropin-releasing hormone-induced luteinizing hormone secretion than does adolescent puberty.