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.

Kisspeptin deficiency leads to abnormal adrenal glands and excess steroid hormone secretion.

Knockout mice for the kisspeptin receptor, Kiss1r (Kiss1r-/-) and its ligand kisspeptin, Kiss1 (Kiss1-/-) replicate the phenotype of isolated hypogonadotropic hypogonadism (IHH) associated with variants of these genes in humans. A recent report suggests that kisspeptin may be involved in human fetal adrenocortical development and function. Herein, we characterized the adrenal function and morphology in Kiss1-/- mice that do not go through normal puberty. Two fetal markers were expressed in eosinophilic cells potentially derived from the X-zone that should disappear at puberty in male mice and during the first pregnancy in female animals. Although the hypercorticosteronism observed in Kiss1-/- females corrected overtime, hyperaldosteronism persisted at 14 months and correlated with the overexpression of Star. To determine if KISS1 and KISS1R genes are involved in the development of primary aldosteronism (PA) and hypercortisolism [Cushing's syndrome (CS)] in humans, we sequenced these 2 genes in 65 patients with PA and/or CS. Interestingly, a patient with CS presented with a germline KISS1 variant (p.H90D, rs201073751). We also found three rare variants in the KISS1R gene in three patients with PA: p.C95W (rs141767649), p.A189T (rs73507527) and p.R229R (rs115335009). The two missense variants have been previously associated with IHH. Our findings suggest that KISS1 may play a role in adrenal function in mice and possibly adrenocortical steroid hormone secretion in humans, beyond its recently described role in human fetal adrenocortical development.

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.

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.

Tachykinin Signaling Is Required for Induction of the Preovulatory Luteinizing Hormone Surge and Normal Luteinizing Hormone Pulses.

Tachykinins (neurokinin A [NKA], neurokinin B [NKB], and substance P [SP]) are important components of the neuroendocrine control of reproduction by direct stimulation of Kiss1 neurons to control GnRH pulsatility, which is essential for reproduction. Despite this role of tachykinins in successful reproduction, knockout (KO) mice for Tac1 (NKA/SP) and Tac2 (NKB) genes are fertile, resembling the phenotype of human patients bearing NKB signaling mutations, who often reverse their hypogonadal phenotype. This suggests the existence of compensatory mechanisms among the different tachykinin ligand-receptor systems to maintain reproduction in the absence of one of them. In order to test this hypothesis, we generated complete tachykinin-deficient mice (Tac1/Tac2KO). Male mice displayed delayed puberty onset and decreased luteinizing hormone (LH) pulsatility (frequency and amplitude of LH pulses) but preserved fertility. However, females did not show signs of puberty onset (first estrus) within 45 days after vaginal opening, they displayed a low frequency (but normal amplitude) of LH pulses, and 80% of them remained infertile. Further evaluation identified a complete absence of the preovulatory LH surge in Tac1/Tac2KO females as well as in wild-type females treated with NKB or SP receptor antagonists. These data confirmed a fundamental role of tachykinins in the timing of puberty onset and LH pulsatility and uncovered a role of tachykinin signaling in facilitation of the preovulatory LH surge. Overall, these findings indicate that tachykinin signaling plays a dominant role in the control of ovulation, with potential implications as a pathogenic mechanism and a therapeutic target to improve reproductive outcomes in women with ovulation impairments.

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.

Safety Evaluation of KP-10 (Metastin 45-54) Following once Daily Intravenous Administration for 14 Days in Dog.

Kisspeptin-10 (previously referred as metastin 45-54), an active fragment of the endogenous full-length kisspeptin-145, is a potential therapeutic agent for reproductive disorders such as infertility, amenorrhea, and pubertal delay. A safety evaluation of KP-10 was conducted in dogs at the doses of 30, 100, and 1,000 µg/kg, given once daily intravenously for 14 days with a 14-day recovery period. There were no overt signs of drug-related toxicity observed in clinical signs, body weights, food consumption, clinical pathology, histopathology, urinalysis, electrocardiogram, or respiratory rate. Due to very rapid clearance of the peptide, luteinizing hormone (LH) levels were measured as a surrogate marker to demonstrate KP-10 exposure. The LH response reached a maximum concentration at 5 minutes post-dose and remained relatively unchanged for at least 30 minutes after dosing with no gender effect. LH concentrations on Day 1 were generally greater than on day 14. Vaginal cytology results indicated all dogs were in anestrous throughout the dosing period. There were also no KP-10-related findings observed in recovery animals on Day 29. In conclusion, KP-10 demonstrated favorable safety profile in dog where 1,000 µg/kg dose was considered as a no-observed-adverse-effect level dose when administered IV once daily for 14 days.

Large-scale meta-analysis highlights the hypothalamic-pituitary-gonadal axis in the genetic regulation of menstrual cycle length.

The normal menstrual cycle requires a delicate interplay between the hypothalamus, pituitary and ovary. Therefore, its length is an important indicator of female reproductive health. Menstrual cycle length has been shown to be partially controlled by genetic factors, especially in the follicle-stimulating hormone beta-subunit (FSHB) locus. A genome-wide association study meta-analysis of menstrual cycle length in 44 871 women of European ancestry confirmed the previously observed association with the FSHB locus and identified four additional novel signals in, or near, the GNRH1, PGR, NR5A2 and INS-IGF2 genes. These findings not only confirm the role of the hypothalamic-pituitary-gonadal axis in the genetic regulation of menstrual cycle length but also highlight potential novel local regulatory mechanisms, such as those mediated by IGF2.

DLG2 variants in patients with pubertal disorders.

PURPOSE: Impaired function of gonadotropin-releasing hormone (GnRH) neurons can cause a phenotypic spectrum ranging from delayed puberty to isolated hypogonadotropic hypogonadism (IHH). We sought to identify a new genetic etiology for these conditions. METHODS: Exome sequencing was performed in an extended family with autosomal dominant, markedly delayed puberty. The effects of the variant were studied in a GnRH neuronal cell line. Variants in the same gene were sought in a large cohort of individuals with IHH. RESULTS: We identified a rare missense variant (F900V) in DLG2 (which encodes PSD-93) that cosegregated with the delayed puberty. The variant decreased GnRH expression in vitro. PSD-93 is an anchoring protein of NMDA receptors, a type of glutamate receptor that has been implicated in the control of puberty in laboratory animals. The F900V variant impaired the interaction between PSD-93 and a known binding partner, Fyn, which phosphorylates NMDA receptors. Variants in DLG2 that also decreased GnRH expression were identified in three unrelated families with IHH. CONCLUSION: The findings indicate that variants in DLG2/PSD-93 cause autosomal dominant delayed puberty and may also contribute to IHH. The findings also suggest that the pathogenesis involves impaired NMDA receptor signaling and consequently decreased GnRH secretion.

Different Cerebellar Ataxia Phenotypes Associated with Mutations of the PNPLA6 Gene in Brazilian Patients with Recessive Ataxias.

Autosomal recessive cerebellar ataxias (ARCAs) represent a heterogeneous group of inherited disorders. The association of early-onset cerebellar ataxia with hypogonadotropic hypogonadism is related to two syndromes, known as Gordon Holmes syndrome (GHS-ataxia and pyramidal signs with hypogonadotropic hypogonadism) and Boucher-Neuhäuser syndrome (BNS-ataxia with chorioretinal dystrophy). Mutations in the PNPLA6 gene have been identified as the cause of hereditary spastic paraplegia and complex forms of ataxia associated with retinal and endocrine manifestations. We reported two Brazilian patients with sporadic, progressive cerebellar ataxia, associated with hypogonadotropic hypogonadism, in whom the GHS and BNS were confirmed by the demonstration of compound heterozygote mutations in the PNPLA6 gene. Genetic analysis of the patient 1 revealed compound heterozygous mutations, one allele in exon 34 and the other allele in exon 29. Genetic exam of the patient 2 also demonstrated compound heterozygous mutations. Three were novel mutations. The missense mutation c.3373G> A, found in the BNS patient, was previously related to Oliver-McFarlane syndrome. These different mutations in this gene suggest a complex phenotype associated disease spectrum.

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.

Ataxia and hypogonadism caused by the loss of ubiquitin ligase activity of the U box protein CHIP.

Gordon Holmes syndrome (GHS) is a rare Mendelian neurodegenerative disorder characterized by ataxia and hypogonadism. Recently, it was suggested that disordered ubiquitination underlies GHS though the discovery of exome mutations in the E3 ligase RNF216 and deubiquitinase OTUD4. We performed exome sequencing in a family with two of three siblings afflicted with ataxia and hypogonadism and identified a homozygous mutation in STUB1 (NM_005861) c.737C→T, p.Thr246Met, a gene that encodes the protein CHIP (C-terminus of HSC70-interacting protein). CHIP plays a central role in regulating protein quality control, in part through its ability to function as an E3 ligase. Loss of CHIP function has long been associated with protein misfolding and aggregation in several genetic mouse models of neurodegenerative disorders; however, a role for CHIP in human neurological disease has yet to be identified. Introduction of the Thr246Met mutation into CHIP results in a loss of ubiquitin ligase activity measured directly using recombinant proteins as well as in cell culture models. Loss of CHIP function in mice resulted in behavioral and reproductive impairments that mimic human ataxia and hypogonadism. We conclude that GHS can be caused by a loss-of-function mutation in CHIP. Our findings further highlight the role of disordered ubiquitination and protein quality control in the pathogenesis of neurodegenerative disease and demonstrate the utility of combining whole-exome sequencing with molecular analyses and animal models to define causal disease polymorphisms.

Mutations in FGF17, IL17RD, DUSP6, SPRY4, and FLRT3 are identified in individuals with congenital hypogonadotropic hypogonadism.

Congenital hypogonadotropic hypogonadism (CHH) and its anosmia-associated form (Kallmann syndrome [KS]) are genetically heterogeneous. Among the >15 genes implicated in these conditions, mutations in FGF8 and FGFR1 account for ~12% of cases; notably, KAL1 and HS6ST1 are also involved in FGFR1 signaling and can be mutated in CHH. We therefore hypothesized that mutations in genes encoding a broader range of modulators of the FGFR1 pathway might contribute to the genetics of CHH as causal or modifier mutations. Thus, we aimed to (1) investigate whether CHH individuals harbor mutations in members of the so-called "FGF8 synexpression" group and (2) validate the ability of a bioinformatics algorithm on the basis of protein-protein interactome data (interactome-based affiliation scoring [IBAS]) to identify high-quality candidate genes. On the basis of sequence homology, expression, and structural and functional data, seven genes were selected and sequenced in 386 unrelated CHH individuals and 155 controls. Except for FGF18 and SPRY2, all other genes were found to be mutated in CHH individuals: FGF17 (n = 3 individuals), IL17RD (n = 8), DUSP6 (n = 5), SPRY4 (n = 14), and FLRT3 (n = 3). Independently, IBAS predicted FGF17 and IL17RD as the two top candidates in the entire proteome on the basis of a statistical test of their protein-protein interaction patterns to proteins known to be altered in CHH. Most of the FGF17 and IL17RD mutations altered protein function in vitro. IL17RD mutations were found only in KS individuals and were strongly linked to hearing loss (6/8 individuals). Mutations in genes encoding components of the FGF pathway are associated with complex modes of CHH inheritance and act primarily as contributors to an oligogenic genetic architecture underlying CHH.

Ataxia, dementia, and hypogonadotropism caused by disordered ubiquitination.

BACKGROUND: The combination of ataxia and hypogonadism was first described more than a century ago, but its genetic basis has remained elusive. METHODS: We performed whole-exome sequencing in a patient with ataxia and hypogonadotropic hypogonadism, followed by targeted sequencing of candidate genes in similarly affected patients. Neurologic and reproductive endocrine phenotypes were characterized in detail. The effects of sequence variants and the presence of an epistatic interaction were tested in a zebrafish model. RESULTS: Digenic homozygous mutations in RNF216 and OTUD4, which encode a ubiquitin E3 ligase and a deubiquitinase, respectively, were found in three affected siblings in a consanguineous family. Additional screening identified compound heterozygous truncating mutations in RNF216 in an unrelated patient and single heterozygous deleterious mutations in four other patients. Knockdown of rnf216 or otud4 in zebrafish embryos induced defects in the eye, optic tectum, and cerebellum; combinatorial suppression of both genes exacerbated these phenotypes, which were rescued by nonmutant, but not mutant, human RNF216 or OTUD4 messenger RNA. All patients had progressive ataxia and dementia. Neuronal loss was observed in cerebellar pathways and the hippocampus; surviving hippocampal neurons contained ubiquitin-immunoreactive intranuclear inclusions. Defects were detected at the hypothalamic and pituitary levels of the reproductive endocrine axis. CONCLUSIONS: The syndrome of hypogonadotropic hypogonadism, ataxia, and dementia can be caused by inactivating mutations in RNF216 or by the combination of mutations in RNF216 and OTUD4. These findings link disordered ubiquitination to neurodegeneration and reproductive dysfunction and highlight the power of whole-exome sequencing in combination with functional studies to unveil genetic interactions that cause disease. (Funded by the National Institutes of Health and others.).

TACR3 mutations disrupt NK3R function through distinct mechanisms in GnRH-deficient patients.

Neurokinin B (NKB) and its G-protein-coupled receptor, NK3R, have been implicated in the neuroendocrine control of GnRH release; however, little is known about the structure-function relationship of this ligand-receptor pair. Moreover, loss-of-function NK3R mutations cause GnRH deficiency in humans. Using missense mutations in NK3R we previously identified in patients with GnRH deficiency, we demonstrate that Y256H and Y315C NK3R mutations in the fifth and sixth transmembrane domains (TM5 and TM6), resulted in reduced whole-cell (79.3±7.2%) or plasma membrane (67.3±7.3%) levels, respectively, compared with wild-type (WT) NK3R, with near complete loss of inositol phosphate (IP) signaling, implicating these domains in receptor trafficking, processing, and/or stability. We further demonstrate in a FRET-based assay that R295S NK3R, in the third intracellular loop (IL3), bound NKB but impaired dissociation of Gq-protein subunits from the receptor compared with WT NK3R, which showed a 10.0 ± 1.3% reduction in FRET ratios following ligand binding, indicating activation of Gq-protein signaling. Interestingly, R295S NK3R, identified in the heterozygous state in a GnRH-deficient patient, also interfered with dissociation of G proteins and IP signaling from wild-type NK3R, indicative of dominant-negative effects. Collectively, our data illustrate roles for TM5 and TM6 in NK3R trafficking and ligand binding and for IL3 in NK3R signaling.

A genetic basis for functional hypothalamic amenorrhea.

BACKGROUND: Functional hypothalamic amenorrhea is a reversible form of gonadotropin-releasing hormone (GnRH) deficiency commonly triggered by stressors such as excessive exercise, nutritional deficits, or psychological distress. Women vary in their susceptibility to inhibition of the reproductive axis by such stressors, but it is unknown whether this variability reflects a genetic predisposition to hypothalamic amenorrhea. We hypothesized that mutations in genes involved in idiopathic hypogonadotropic hypogonadism, a congenital form of GnRH deficiency, are associated with hypothalamic amenorrhea. METHODS: We analyzed the coding sequence of genes associated with idiopathic hypogonadotropic hypogonadism in 55 women with hypothalamic amenorrhea and performed in vitro studies of the identified mutations. RESULTS: Six heterozygous mutations were identified in 7 of the 55 patients with hypothalamic amenorrhea: two variants in the fibroblast growth factor receptor 1 gene FGFR1 (G260E and R756H), two in the prokineticin receptor 2 gene PROKR2 (R85H and L173R), one in the GnRH receptor gene GNRHR (R262Q), and one in the Kallmann syndrome 1 sequence gene KAL1 (V371I). No mutations were found in a cohort of 422 controls with normal menstrual cycles. In vitro studies showed that FGFR1 G260E, FGFR1 R756H, and PROKR2 R85H are loss-of-function mutations, as has been previously shown for PROKR2 L173R and GNRHR R262Q. CONCLUSIONS: Rare variants in genes associated with idiopathic hypogonadotropic hypogonadism are found in women with hypothalamic amenorrhea, suggesting that these mutations may contribute to the variable susceptibility of women to the functional changes in GnRH secretion that characterize hypothalamic amenorrhea. Our observations provide evidence for the role of rare variants in common multifactorial disease. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others; ClinicalTrials.gov number, NCT00494169.).

Heparan sulfate 6-O-sulfotransferase 1, a gene involved in extracellular sugar modifications, is mutated in patients with idiopathic hypogonadotrophic hypogonadism.

Neuronal development is the result of a multitude of neural migrations, which require extensive cell-cell communication. These processes are modulated by extracellular matrix components, such as heparan sulfate (HS) polysaccharides. HS is molecularly complex as a result of nonrandom modifications of the sugar moieties, including sulfations in specific positions. We report here mutations in HS 6-O-sulfotransferase 1 (HS6ST1) in families with idiopathic hypogonadotropic hypogonadism (IHH). IHH manifests as incomplete or absent puberty and infertility as a result of defects in gonadotropin-releasing hormone neuron development or function. IHH-associated HS6ST1 mutations display reduced activity in vitro and in vivo, suggesting that HS6ST1 and the complex modifications of extracellular sugars are critical for normal development in humans. Genetic experiments in Caenorhabditis elegans reveal that HS cell-specifically regulates neural branching in vivo in concert with other IHH-associated genes, including kal-1, the FGF receptor, and FGF. These findings are consistent with a model in which KAL1 can act as a modulatory coligand with FGF to activate the FGF receptor in an HS-dependent manner.

Lack of a genetic risk continuum between pubertal timing in the general population and idiopathic hypogonadotropic hypogonadism.

Pubertal timing is a highly heritable trait in the general population. Recently, a large-scale exome-wide association study has implicated rare variants in six genes (KDM4C, MC3R, MKRN3, PDE10A, TACR3, and ZNF483) as genetic determinants of pubertal timing within the general population. Two of the genes (TACR3, MKRN3) are already implicated in extreme disorders of pubertal timing. This observation suggests that there may be a pervasive "genetic risk continuum" wherein genes that govern pubertal timing in the general population, by extension, may also be causal for rare Mendelian disorders of pubertal timing. Hence, we hypothesized that the four novel genes linked to pubertal timing in the population will also contribute to idiopathic hypogonadotropic hypogonadism (IHH), a genetic disorder characterized by absent puberty. Exome sequencing data from 1322 unrelated IHH probands were reviewed for rare sequence variants (RSVs) (minor allele frequency bins: <1%; <0.1%; <0.01%) in the six genes linked to puberty in the general population. A gene-based rare variant association testing (RVAT) was performed between the IHH cohort and a reference public genomic sequences repository-the Genome Aggregation Database (gnomAD). As expected, RVAT analysis showed that RSVs in TACR3, a known IHH gene, were significantly enriched in the IHH cohort compared to gnomAD cohort across all three MAF bins. However, RVAT analysis of the remaining five genes failed to show any RSV enrichment in the IHH cohort across all MAF bins. Our findings argue strongly against a pervasive genetic risk continuum between pubertal timing in the general population and extreme pubertal phenotypes. The biologic basis of such distinct genetic architectures' merits further evaluation.

Expanding the Spectrum of Endocrine Abnormalities Associated with SOX11-related Disorders.

CONTEXT: SOX11 variants cause Coffin-Siris Syndrome (CSS), characterized by developmental delay, hypogonadotropic hypogonadism (HH), skeletal and facial defects. OBJECTIVE: To examine the contribution of SOX11 variants to the pathogenesis of Idiopathic Hypogonadotropic Hypogonadism (IHH), a disorder caused by hypothalamic GnRH deficiency. SETTING: The Reproductive Endocrine Unit and the Pediatric Endocrinology Division, Massachusetts General Hospital. PATIENTS OR OTHER PARTICIPANTS: A cohort of 1810 unrelated IHH probands. INTERVENTIONS: Exome sequencing data from the entire cohort were examined for SOX11 rare single nucleotide variants (SNVs) [minor allele frequency in the gnomAD database <0.1%]. Rare SOX11 variant association testing was performed between the IHH and gnomAD population. Phenotyping of individuals harboring pathogenic/likely pathogenic SNVs (determined by the ACMG criteria) was performed. MAIN OUTCOMES/RESULTS: Four pathogenic SOX11 SNVs were identified in 5 IHH probands. The IHH cohort was enriched for SOX11 protein truncating SNVs (frameshift/nonsense) across the entire protein (2 SNVs in 3 IHH cases [p.S303X (de-novo); p.S345Afs*13]; p 0.0004981) and for SOX11 missense SNVs within the SOX11-high-mobility group (HMG) domain (2 SNVs in 2 IHH cases p.G84D[de-novo]; p.P114S; p=0.00313922). The phenotypic spectrum of SOX11 variant carriers revealed additional endocrine defects including anosmic and normosmic forms of IHH, growth-hormone deficiency, pituitary and hypothalamic structural defects, and hypothyroidism. A pathogenic SOX11 SNV was also identified in a patient with functional HH (FHH, p.R100Q). CSS-associated features were present in 4/5 probands. CONCLUSIONS: Deleterious SOX11 variants cause IHH and other pituitary hormone deficiencies, suggesting that the human SOX11-associated disorder may stem from both hypothalamic and pituitary level defects.