RESUMEN
BACKGROUND/OBJECTIVES: This study aims to elucidate the genetic causes of congenital hypogonadotropic hypogonadism (CHH), a rare genetic disorder resulting in GnRH deficiency, in six families from Pakistan. METHODS: Eighteen DNA samples from six families underwent genome sequencing followed by standard evaluation for pathogenic single nucleotide variants (SNVs) and small indels. All families were subsequently analyzed for pathogenic copy number variants (CNVs) using CoverageMaster. RESULTS: Novel pathogenic homozygous SNVs in known CHH genes were identified in four families: two families with variants in GNRHR, and two others harboring KISS1R variants. Subsequent investigation of CNVs in the remaining two families identified novel unique large deletions in ANOS1. CONCLUSION: A combined, systematic analysis of single nucleotide and CNVs helps to improve the diagnostic yield for variants in patients with CHH.
Asunto(s)
Variaciones en el Número de Copia de ADN , Hipogonadismo , Linaje , Polimorfismo de Nucleótido Simple , Humanos , Hipogonadismo/genética , Pakistán , Masculino , Femenino , Receptores de Kisspeptina-1/genética , Secuenciación Completa del Genoma , Receptores LHRH/genética , Adulto , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso , Proteínas de la Matriz ExtracelularRESUMEN
Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic disorder characterized by infertility and the absence of puberty. Defects in GnRH neuron migration or altered GnRH secretion and/or action lead to a severe gonadotropin-releasing hormone (GnRH) deficiency. Given the close developmental association of GnRH neurons with the olfactory primary axons, CHH is often associated with anosmia or hyposmia, in which case it is defined as Kallmann syndrome (KS). The genetics of CHH are heterogeneous, and >40 genes are involved either alone or in combination. Several CHH-related genes controlling GnRH ontogeny encode proteins containing fibronectin-3 (FN3) domains, which are important for brain and neural development. Therefore, we hypothesized that defects in other FN3-superfamily genes would underlie CHH. Next-generation sequencing was performed for 240 CHH unrelated probands and filtered for rare, protein-truncating variants (PTVs) in FN3-superfamily genes. Compared to gnomAD controls the CHH cohort was statistically enriched for PTVs in neuron-derived neurotrophic factor (NDNF) (p = 1.40 × 10-6). Three heterozygous PTVs (p.Lys62∗, p.Tyr128Thrfs∗55, and p.Trp469∗, all absent from the gnomAD database) and an additional heterozygous missense mutation (p.Thr201Ser) were found in four KS probands. Notably, NDNF is expressed along the GnRH neuron migratory route in both mouse embryos and human fetuses and enhances GnRH neuron migration. Further, knock down of the zebrafish ortholog of NDNF resulted in altered GnRH migration. Finally, mice lacking Ndnf showed delayed GnRH neuron migration and altered olfactory axonal projections to the olfactory bulb; both results are consistent with a role of NDNF in GnRH neuron development. Altogether, our results highlight NDNF as a gene involved in the GnRH neuron migration implicated in KS.
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Movimiento Celular , Hipogonadismo/congénito , Hipogonadismo/genética , Mutación , Factores de Crecimiento Nervioso/genética , Neuronas/patología , Adolescente , Animales , Estudios de Cohortes , Femenino , Heterocigoto , Humanos , Hipogonadismo/patología , Masculino , Ratones , Ratones Noqueados , Factores de Crecimiento Nervioso/fisiología , Neuronas/metabolismo , Linaje , Pez CebraRESUMEN
Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic disease characterized by absent puberty and infertility due to GnRH deficiency, and is often associated with anosmia [Kallmann syndrome (KS)]. The genetic etiology of CHH is heterogeneous, and more than 30 genes have been implicated in approximately 50% of patients with CHH. We hypothesized that genes encoding axon-guidance proteins containing fibronectin type-III (FN3) domains (similar to ANOS1, the first gene associated with KS), are mutated in CHH. We performed whole-exome sequencing in a cohort of 133 CHH probands to test this hypothesis, and identified rare sequence variants (RSVs) in genes encoding for the FN3-domain encoding protein deleted in colorectal cancer (DCC) and its ligand Netrin-1 (NTN1). In vitro studies of these RSVs revealed altered intracellular signaling associated with defects in cell morphology, and confirmed five heterozygous DCC mutations in 6 probands-5 of which presented as KS. Two KS probands carry heterozygous mutations in both DCC and NTN1 consistent with oligogenic inheritance. Further, we show that Netrin-1 promotes migration in immortalized GnRH neurons (GN11 cells). This study implicates DCC and NTN1 mutations in the pathophysiology of CHH consistent with the role of these two genes in the ontogeny of GnRH neurons in mice.
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Receptor DCC/genética , Hipogonadismo/genética , Netrina-1/genética , Adulto , Estudios de Cohortes , Receptor DCC/metabolismo , Femenino , Dominio de Fibronectina del Tipo III , Hormona Liberadora de Gonadotropina/deficiencia , Humanos , Hipogonadismo/metabolismo , Hipogonadismo/patología , Masculino , Mutación , Netrina-1/metabolismo , Neuronas/metabolismo , Neuronas/patología , Linaje , Secuenciación del ExomaRESUMEN
PURPOSE: Congenital hypogonadotropic hypogonadism (CHH) is a rare disorder resulting in absent puberty and infertility. The genetic architecture is complex with multiple loci involved, variable expressivity, and incomplete penetrance. The majority of cases are sporadic, consistent with a disease affecting fertility. The current study aims to investigate mosaicism as a genetic mechanism for CHH, focusing on de novo rare variants in CHH genes. METHODS: We evaluated 60 trios for de novo rare sequencing variants (RSV) in known CHH genes using exome sequencing. Potential mosaicism was suspected among RSVs with altered allelic ratios and confirmed using customized ultradeep sequencing (UDS) in multiple tissues. RESULTS: Among the 60 trios, 10 probands harbored de novo pathogenic variants in CHH genes. Custom UDS demonstrated that three of these de novo variants were in fact postzygotic mosaicism-two in FGFR1 (p.Leu630Pro and p.Gly348Arg), and one in CHD7 (p.Arg2428*). Statistically significant variation across multiple tissues (DNA from blood, buccal, hair follicle, urine) confirmed their mosaic nature. CONCLUSIONS: We identified a significant number of de novo pathogenic variants in CHH of which a notable number (3/10) exhibited mosaicism. This report of postzygotic mosaicism in CHH patients provides valuable information for accurate genetic counseling.
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Hipogonadismo , Infertilidad , Asesoramiento Genético , Humanos , Hipogonadismo/genética , Mosaicismo , Secuenciación del ExomaRESUMEN
ß-Klotho (encoded by Klb) is an obligate coreceptor, mediating both fibroblast growth factor (FGF)15 and FGF21 signaling. Klb-/- mice are refractory to metabolic FGF15 and FGF21 action and exhibit derepressed (increased) bile acid (BA) synthesis. Here, we deeply phenotyped male Klb-/- mice on a pure C57BL/6J genetic background, fed a chow diet focusing on metabolic aspects. This aims to better understand the physiological consequences of concomitant FGF15 and FGF21 signaling deficiency, in particular on the gut-liver axis. Klb-/- mice present permanent growth restriction independent of adiposity and energy balance. Klb-/- mice also exhibit few changes in carbohydrate metabolism, combining normal gluco-tolerance, insulin sensitivity, and fasting response with increased gluconeogenic capacity and decreased glycogen mobilization. Livers of Klb-/- mice reveal pathologic features, including a proinflammatory status and initiation of fibrosis. These defects are associated to a massive shift in BA composition in the enterohepatic system and blood circulation featured by a large excess of microbiota-derived deoxycholic acid, classically known for its genotoxicity in the gastrointestinal tract. In conclusion, ß-Klotho is a gatekeeper of hepatic integrity through direct action (mediating FGF21 anti-inflammatory signaling) and indirect mechanisms (mediating FGF15 signaling that maintains BA level and composition).
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Ácidos y Sales Biliares/metabolismo , Peso Corporal/fisiología , Tracto Gastrointestinal/metabolismo , Cirrosis Hepática/metabolismo , Hígado/metabolismo , Proteínas de la Membrana/metabolismo , Adiposidad/genética , Animales , Metabolismo Energético/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Gluconeogénesis/fisiología , Cuerpos Cetónicos/sangre , Proteínas Klotho , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Transducción de Señal/fisiologíaRESUMEN
PURPOSE: Congenital hypogonadotropic hypogonadism (CHH), a rare genetic disease caused by gonadotropin-releasing hormone deficiency, can also be part of complex syndromes (e.g., CHARGE syndrome). CHD7 mutations were reported in 60% of patients with CHARGE syndrome, and in 6% of CHH patients. However, the definition of CHD7 mutations was variable, and the associated CHARGE signs in CHH were not systematically examined. METHODS: Rare sequencing variants (RSVs) in CHD7 were identified through exome sequencing in 116 CHH probands, and were interpreted according to American College of Medical Genetics and Genomics guidelines. Detailed phenotyping was performed in CHH probands who were positive for CHD7 RSVs, and genotype-phenotype correlations were evaluated. RESULTS: Of the CHH probands, 16% (18/116) were found to harbor heterozygous CHD7 RSVs, and detailed phenotyping was performed in 17 of them. Of CHH patients with pathogenic or likely pathogenic CHD7 variants, 80% (4/5) were found to exhibit multiple CHARGE features, and 3 of these patients were reclassified as having CHARGE syndrome. In contrast, only 8% (1/12) of CHH patients with nonpathogenic CHD7 variants exhibited multiple CHARGE features (P = 0.01). CONCLUSION: Pathogenic or likely pathogenic CHD7 variants rarely cause isolated CHH. Therefore a detailed clinical investigation is indicated to clarify the diagnosis (CHH versus CHARGE) and to optimize clinical management.
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Síndrome CHARGE/genética , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Hipogonadismo/genética , Síndrome CHARGE/diagnóstico , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Familia , Femenino , Estudios de Asociación Genética , Variación Genética/genética , Heterocigoto , Humanos , Masculino , Mutación , Linaje , Fenotipo , Análisis de Secuencia de ADNRESUMEN
Acetyl-CoA carboxylase 2 (ACC2) is an isoform of ACC functioning as a negative regulator of fatty acid ß-oxidation. Spot14, a thyroid hormone responsive protein, and Mig12, a Spot14 paralog, have recently been identified as regulators of fatty acid synthesis targeting ACC1, a distinctive subtype of ACC. Here, we examined whether Spot14/Mig12 modulates ACC2. Nanoscale protein topography mapped putative protein-protein interactions between purified human Spot14/Mig12 and ACC2, validated by functional assays. Human ACC2 displayed consistent enzymatic activity, and homogeneous particle distribution was probed by atomic force microscopy. Citrate-induced polymerization and enzymatic activity of ACC2 were restrained by the addition of the recombinant Spot14/Mig12 heterocomplex but only partially by the oligo-heterocomplex, demonstrating that the heterocomplex is a designated metabolic inhibitor of human ACC2. Moreover, Spot14/Mig12 demonstrated a sequestering role preventing an initial ACC2 nucleation step during filamentous polymer formation. Thus, the Spot14/Mig12 heterocomplex controls human ACC2 polymerization and catalytic function, emerging as a previously unrecognized molecular regulator in catalytic lipid metabolism.
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Acetil-CoA Carboxilasa/química , Acetil-CoA Carboxilasa/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Catálisis , Ácidos Grasos/metabolismo , Humanos , Microscopía de Fuerza Atómica , Oxidación-Reducción , Unión ProteicaRESUMEN
The nitric oxide (NO) signaling pathway in hypothalamic neurons plays a key role in the regulation of the secretion of gonadotropin-releasing hormone (GnRH), which is crucial for reproduction. We hypothesized that a disruption of neuronal NO synthase (NOS1) activity underlies some forms of hypogonadotropic hypogonadism. Whole-exome sequencing was performed on a cohort of 341 probands with congenital hypogonadotropic hypogonadism to identify ultrarare variants in NOS1. The activity of the identified NOS1 mutant proteins was assessed by their ability to promote nitrite and cGMP production in vitro. In addition, physiological and pharmacological characterization was carried out in a Nos1-deficient mouse model. We identified five heterozygous NOS1 loss-of-function mutations in six probands with congenital hypogonadotropic hypogonadism (2%), who displayed additional phenotypes including anosmia, hearing loss, and intellectual disability. NOS1 was found to be transiently expressed by GnRH neurons in the nose of both humans and mice, and Nos1 deficiency in mice resulted in dose-dependent defects in sexual maturation as well as in olfaction, hearing, and cognition. The pharmacological inhibition of NO production in postnatal mice revealed a critical time window during which Nos1 activity shaped minipuberty and sexual maturation. Inhaled NO treatment at minipuberty rescued both reproductive and behavioral phenotypes in Nos1-deficient mice. In summary, lack of NOS1 activity led to GnRH deficiency associated with sensory and intellectual comorbidities in humans and mice. NO treatment during minipuberty reversed deficits in sexual maturation, olfaction, and cognition in Nos1 mutant mice, suggesting a potential therapy for humans with NO deficiency.
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Hipogonadismo , Óxido Nítrico , Animales , Cognición , Hormona Liberadora de Gonadotropina/genética , Hormona Liberadora de Gonadotropina/metabolismo , Humanos , Hipogonadismo/complicaciones , Hipogonadismo/congénito , Hipogonadismo/genética , Ratones , Proteínas Mutantes , Mutación/genética , Óxido Nítrico Sintasa de Tipo I/genética , NitritosRESUMEN
Genomic perturbations that challenge normal signaling at the pluripotent stage may trigger unforeseen ontogenic aberrancies. Anticipatory systems biology identification of transcriptome landscapes that underlie latent phenotypes would offer molecular diagnosis before the onset of symptoms. The purpose of this study was to assess the impact of calreticulin-deficient embryonic stem cell transcriptomes on molecular functions and physiological systems. Bioinformatic surveillance of calreticulin-null stem cells, a monogenic insult model, diagnosed a disruption in transcriptome dynamics, which re-prioritized essential cellular functions. Calreticulin-calibrated signaling axes were uncovered, and network-wide cartography of undifferentiated stem cell transcripts suggested cardiac manifestations. Calreticulin-deficient stem cell-derived cardiac cells verified disorganized sarcomerogenesis, mitochondrial paucity, and cytoarchitectural aberrations to validate calreticulin-dependent network forecasts. Furthermore, magnetic resonance imaging and histopathology detected a ventricular septal defect, revealing organogenic manifestation of calreticulin deletion. Thus, bioinformatic deciphering of a primordial calreticulin-deficient transcriptome decoded at the pluripotent stem cell stage a reconfigured multifunctional molecular registry to anticipate predifferentiation susceptibility toward abnormal cardiophenotype.
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Calreticulina/metabolismo , Células Madre Embrionarias/metabolismo , Miocardio/metabolismo , Animales , Calreticulina/deficiencia , Muerte Celular , Diferenciación Celular , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/citología , Perfilación de la Expresión Génica , Ratones , Ratones Noqueados , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Miocardio/citología , FenotipoRESUMEN
In the developing embryo, instructive guidance from the ventral endoderm secures cardiac program induction within the anterolateral mesoderm. Endoderm-guided cardiogenesis, however, has yet to be resolved at the proteome level. Here, through cardiopoietic priming of the endoderm with the reprogramming cytokine tumor necrosis factor alpha (TNFalpha), candidate effectors of embryonic stem cell cardiac differentiation were delineated by comparative proteomics. Differential two-dimensional gel electrophoretic mapping revealed that more than 75% of protein species increased >1.5-fold in the TNFalpha-primed versus unprimed endodermal secretome. Protein spot identification by linear ion trap quadrupole (LTQ) tandem mass spectrometry (MS/MS) and validation by shotgun LTQ-Fourier transform MS/MS following multidimensional chromatography mapped 99 unique proteins from 153 spot assignments. A definitive set of 48 secretome proteins was deduced by iterative bioinformatic screening using algorithms for detection of canonical and noncanonical indices of secretion. Protein-protein interaction analysis, in conjunction with respective expression level changes, revealed a nonstochastic TNFalpha-centric secretome network with a scale-free hierarchical architecture. Cardiovascular development was the primary developmental function of the resolved TNFalpha-anchored network. Functional cooperativity of the derived cardioinductive network was validated through direct application of the TNFalpha-primed secretome on embryonic stem cells, potentiating cardiac commitment and sarcomerogenesis. Conversely, inhibition of primary network hubs negated the procardiogenic effects of TNFalpha priming. Thus, proteomic cartography establishes a systems biology framework for the endodermal secretome network guiding stem cell cardiopoiesis.
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Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Corazón Fetal/citología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Células Madre Embrionarias/citología , Endodermo/citología , Endodermo/efectos de los fármacos , Endodermo/metabolismo , Corazón Fetal/efectos de los fármacos , Corazón Fetal/embriología , Corazón Fetal/metabolismo , Ratones , Mapeo de Interacción de Proteínas , Proteoma/aislamiento & purificación , Proteómica , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic form of isolated gonadotropin-releasing hormone (GnRH) deficiency caused by mutations in > 30 genes. Fibroblast growth factor receptor 1 (FGFR1) is the most frequently mutated gene in CHH and is implicated in GnRH neuron development and maintenance. We note that a CHH FGFR1 mutation (p.L342S) decreases signaling of the metabolic regulator FGF21 by impairing the association of FGFR1 with ß-Klotho (KLB), the obligate co-receptor for FGF21. We thus hypothesized that the metabolic FGF21/KLB/FGFR1 pathway is involved in CHH Genetic screening of 334 CHH patients identified seven heterozygous loss-of-function KLB mutations in 13 patients (4%). Most patients with KLB mutations (9/13) exhibited metabolic defects. In mice, lack of Klb led to delayed puberty, altered estrous cyclicity, and subfertility due to a hypothalamic defect associated with inability of GnRH neurons to release GnRH in response to FGF21. Peripheral FGF21 administration could indeed reach GnRH neurons through circumventricular organs in the hypothalamus. We conclude that FGF21/KLB/FGFR1 signaling plays an essential role in GnRH biology, potentially linking metabolism with reproduction.
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Factores de Crecimiento de Fibroblastos/metabolismo , Hormona Liberadora de Gonadotropina/metabolismo , Síndrome de Kallmann/genética , Proteínas de la Membrana/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Animales , Células COS , Caenorhabditis elegans/genética , Chlorocebus aethiops , Estudios de Cohortes , Femenino , Factores de Crecimiento de Fibroblastos/genética , Hormona Liberadora de Gonadotropina/genética , Células HEK293 , Humanos , Hipotálamo/metabolismo , Proteínas Klotho , Masculino , Ratones Endogámicos C57BL , Ratones Mutantes , Neuronas/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genéticaRESUMEN
Expression of p16(Ink4a) and p19(Arf) increases with age in both rodent and human tissues. However, whether these tumour suppressors are effectors of ageing remains unclear, mainly because knockout mice lacking p16(Ink4a) or p19(Arf) die early of tumours. Here, we show that skeletal muscle and fat, two tissues that develop early ageing-associated phenotypes in response to BubR1 insufficiency, have high levels of p16(Ink4a) and p19(Arf). Inactivation of p16(Ink4a) in BubR1-insufficient mice attenuates both cellular senescence and premature ageing in these tissues. Conversely, p19(Arf) inactivation exacerbates senescence and ageing in BubR1 mutant mice. Thus, we identify BubR1 insufficiency as a trigger for activation of the Cdkn2a locus in certain mouse tissues, and demonstrate that p16(Ink4a) is an effector and p19(Arf) an attenuator of senescence and ageing in these tissues.
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Envejecimiento/fisiología , Senescencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Tejido Adiposo/anatomía & histología , Tejido Adiposo/metabolismo , Animales , Biomarcadores/metabolismo , Proteínas de Ciclo Celular , Células Cultivadas , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Ojo/anatomía & histología , Ojo/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Genotipo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Serina-Treonina Quinasas/genética , Tasa de Supervivencia , Distribución TisularRESUMEN
Nuclear transport of transcription factors is a critical step in stem cell commitment to a tissue-specific lineage. While it is recognized that nuclear pores are gatekeepers of nucleocytoplasmic exchange, it is unknown how the nuclear transport machinery becomes competent to support genetic reprogramming and cell differentiation. Here, we report the dynamics of nuclear transport factor expression and nuclear pore microanatomy during cardiac differentiation of embryonic stem cells. Cardiac progeny derived from pluripotent stem cells displayed a distinct proteomic profile characterized by the emergence of cardiac-specific proteins. This profile correlated with the nuclear translocation of cardiac transcription factors. The nuclear transport genes, including nucleoporins, importins, exportins, transportins, and Ran-related factors, were globally downregulated at the genomic level, streamlining the differentiation program underlying stem cell-derived cardiogenesis. Establishment of the cardiac molecular phenotype was associated with an increased density of nuclear pores spanning the nuclear envelope. At nanoscale resolution, individual nuclear pores exhibited conformational changes resulting in the expansion of the pore diameter and an augmented probability of conduit occupancy. Thus, embryonic stem cells undergo adaptive remodeling of the nuclear transport infrastructure associated with nuclear translocation of cardiac transcription factors and execution of the cardiogenic program, underscoring the plasticity of the nucleocytoplasmic trafficking machinery in accommodating differentiation requirements.