RESUMO
Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is a congenital condition characterized by aplasia or hypoplasia of the uterus and vagina in women with a 46,XX karyotype. This condition can occur as type I when isolated or as type II when associated with extragenital anomalies including kidney and skeletal abnormalities. The genetic basis of MRKH syndrome remains unexplained and several candidate genes have been proposed to play a role in its etiology, including HNF1B, LHX1 and WNT4. Here, we conducted a microarray analysis of 13 women affected by MRKH syndrome, resulting in the identification of chromosomal changes, including the deletion at 17q12, which contains both HNF1B and LHX1. We focused on HNF1B for further investigation due to its known association with, but unknown etiological role in, MRKH syndrome. We ablated Hnf1b specifically in the epithelium of the Müllerian ducts in mice and found that this caused hypoplastic development of the uterus, as well as kidney anomalies, closely mirroring the MRKH type II phenotype. Using single-cell RNA sequencing of uterine tissue in the Hnf1b-ablated embryos, we analyzed the molecules and pathways downstream of Hnf1b, revealing a dysregulation of processes associated with cell proliferation, migration and differentiation. Thus, we establish that loss of Hnf1b function leads to an MRKH phenotype and generate the first mouse model of MRKH syndrome type II. Our results support the investigation of HNF1B in clinical genetic settings of MRKH syndrome and shed new light on the molecular mechanisms underlying this poorly understood condition in women's reproductive health.
Assuntos
Transtornos 46, XX do Desenvolvimento Sexual , Ductos Paramesonéfricos , Animais , Feminino , Camundongos , Transtornos 46, XX do Desenvolvimento Sexual/genética , Diferenciação Celular , Genômica , Fator 1-beta Nuclear de Hepatócito/genética , HumanosRESUMO
Premature ovarian insufficiency is a common form of female infertility affecting up to 4% of women and characterised by amenorrhea with elevated gonadotropin before the age of 40. Oocytes require controlled DNA breakage and repair for homologous recombination and the maintenance of oocyte integrity. Biallelic disruption of the DNA damage repair gene, Fanconi anemia complementation group A (FANCA), is a common cause of Fanconi anaemia, a syndrome characterised by bone marrow failure, cancer predisposition, physical anomalies and POI. There is ongoing dispute about the role of heterozygous FANCA variants in POI pathogenesis, with insufficient evidence supporting causation. Here, we have identified biallelic FANCA variants in French sisters presenting with POI, including a novel missense variant of uncertain significance and a likely pathogenic deletion that initially evaded detection. Functional studies indicated no discernible effect on DNA damage sensitivity in patient lymphoblasts. These novel FANCA variants add evidence that heterozygous loss of one allele is insufficient to cause DNA damage sensitivity and POI. We propose that intragenic deletions, that are relatively common in FANCA, may be missed without careful analysis, and could explain the presumed causation of heterozygous variants. Accurate variant curation is critical to optimise patient care and outcomes.
Assuntos
Alelos , Proteína do Grupo de Complementação A da Anemia de Fanconi , Insuficiência Ovariana Primária , Humanos , Insuficiência Ovariana Primária/genética , Feminino , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Adulto , Anemia de Fanconi/genética , Anemia de Fanconi/diagnóstico , Irmãos , Heterozigoto , Predisposição Genética para Doença , Linhagem , Mutação/genéticaRESUMO
OBJECTIVES: To investigate the approach taken by clinicians involved in the diagnosis and management of individuals with Differences of Sex Development (DSD), particularly with regard to genomic testing, and identify perceived gaps/strengths/barriers in current practice. DESIGN AND METHODS: An anonymous online survey was developed, with questions exploring demographics, perceptions of genomic testing, availability of genetics services and opinions on the role and utility of genomic testing in DSD. All responses were anonymous. Clinicians involved in the diagnosis and management of individuals with DSD were recruited from relevant societies and departments across Australia and New Zealand. RESULTS: 79 eligible clinicians commenced the survey, with 63 completing it and 16 providing a partial response. The perceived benefit of having a genetic diagnosis for DSD was almost unanimous (97%). Almost half (48%) of respondents reported barriers in genomic testing. 81% of respondents reported they order genomic tests currently. Approaches to genomic testing when faced with four different clinical scenarios varied across respondents. Clinicians perceived genomic testing to be underutilised (median 36 on sliding scale from 0 to 100). CONCLUSIONS: Despite 97% of respondents reporting benefit of a genetic diagnosis for individuals with DSD, this was not reflected throughout the survey with regard to clinical implementation. When faced with clinical scenarios, the recommendations for genomic testing from respondents was much lower, indicating the discrepancy between perception and clinical practice. Genomic testing in the context of DSD is seen as both beneficial and desired, yet there are multiple barriers impacting its integration into standard clinical care.
RESUMO
Premature ovarian insufficiency (POI) is a common cause of infertility in women, characterised by amenorrhea and elevated FSH under the age of 40 years. In some cases, POI is syndromic in association with other features such as sensorineural hearing loss in Perrault syndrome. POI is a heterogeneous disease with over 80 causative genes known so far; however, these explain only a minority of cases. Using whole-exome sequencing (WES), we identified a MRPL50 homozygous missense variant (c.335T > A; p.Val112Asp) shared by twin sisters presenting with POI, bilateral high-frequency sensorineural hearing loss, kidney and heart dysfunction. MRPL50 encodes a component of the large subunit of the mitochondrial ribosome. Using quantitative proteomics and western blot analysis on patient fibroblasts, we demonstrated a loss of MRPL50 protein and an associated destabilisation of the large subunit of the mitochondrial ribosome whilst the small subunit was preserved. The mitochondrial ribosome is responsible for the translation of subunits of the mitochondrial oxidative phosphorylation machinery, and we found patient fibroblasts have a mild but significant decrease in the abundance of mitochondrial complex I. These data support a biochemical phenotype associated with MRPL50 variants. We validated the association of MRPL50 with the clinical phenotype by knockdown/knockout of mRpL50 in Drosophila, which resulted abnormal ovarian development. In conclusion, we have shown that a MRPL50 missense variant destabilises the mitochondrial ribosome, leading to oxidative phosphorylation deficiency and syndromic POI, highlighting the importance of mitochondrial support in ovarian development and function.
Assuntos
Disgenesia Gonadal 46 XX , Perda Auditiva Neurossensorial , Insuficiência Ovariana Primária , Feminino , Humanos , Disgenesia Gonadal 46 XX/genética , Perda Auditiva Neurossensorial/genética , Mitocôndrias/genética , Mutação de Sentido Incorreto , Insuficiência Ovariana Primária/genética , Animais , Drosophila melanogasterRESUMO
Premature ovarian insufficiency (POI) is a leading form of female infertility, characterised by menstrual disturbance and elevated follicle-stimulating hormone before age 40. It is highly heterogeneous with variants in over 80 genes potentially causative, but the majority of cases having no known cause. One gene implicated in POI pathology is TP63. TP63 encodes multiple p63 isoforms, one of which has been shown to have a role in the surveillance of genetic quality in oocytes. TP63 C-terminal truncation variants and N-terminal duplication have been described in association with POI, however, functional validation has been lacking. Here we identify three novel TP63 missense variants in women with nonsyndromic POI, including one in the N-terminal activation domain, one in the C-terminal inhibition domain, and one affecting a unique and poorly understood p63 isoform, TA*p63. Via blue-native page and luciferase reporter assays we demonstrate that two of these variants disrupt p63 dimerization, leading to constitutively active p63 tetramer that significantly increases the transcription of downstream targets. This is the first evidence that TP63 missense variants can cause isolated POI and provides mechanistic insight that TP63 variants cause POI due to constitutive p63 activation and accelerated oocyte loss in the absence of DNA damage.
Assuntos
Insuficiência Ovariana Primária , Fatores de Transcrição , Proteínas Supressoras de Tumor , Feminino , Humanos , Mutação de Sentido Incorreto , Insuficiência Ovariana Primária/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Perrault syndrome is a rare heterogeneous condition characterised by sensorineural hearing loss and premature ovarian insufficiency. Additional neuromuscular pathology is observed in some patients. There are six genes in which variants are known to cause Perrault syndrome; however, these explain only a minority of cases. We investigated the genetic cause of Perrault syndrome in seven affected individuals from five different families, successfully identifying the cause in four patients. This included previously reported and novel causative variants in known Perrault syndrome genes, CLPP and LARS2, involved in mitochondrial proteolysis and mitochondrial translation, respectively. For the first time, we show that pathogenic variants in PEX6 can present clinically as Perrault syndrome. PEX6 encodes a peroxisomal biogenesis factor, and we demonstrate evidence of peroxisomal dysfunction in patient serum. This study consolidates the clinical overlap between Perrault syndrome and peroxisomal disorders, and highlights the need to consider ovarian function in individuals with atypical/mild peroxisomal disorders. The remaining patients had variants in candidate genes such as TFAM, involved in mtDNA transcription, replication, and packaging, and GGPS1 involved in mevalonate/coenzyme Q10 biosynthesis and whose enzymatic product is required for mouse folliculogenesis. This genomic study highlights the diverse molecular landscape of this poorly understood syndrome.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Aminoacil-tRNA Sintetases/genética , Proteínas de Ligação a DNA/genética , Dimetilaliltranstransferase/genética , Endopeptidase Clp/genética , Farnesiltranstransferase/genética , Predisposição Genética para Doença , Geraniltranstransferase/genética , Disgenesia Gonadal 46 XX/genética , Perda Auditiva Neurossensorial/genética , Proteínas Mitocondriais/genética , Fatores de Transcrição/genética , Adolescente , Adulto , Sequência de Bases , Criança , DNA Mitocondrial/genética , Feminino , Expressão Gênica , Disgenesia Gonadal 46 XX/diagnóstico , Disgenesia Gonadal 46 XX/patologia , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Ovário/metabolismo , Ovário/patologia , Linhagem , Peroxissomos/metabolismo , Peroxissomos/patologiaRESUMO
Infertility, a global problem affecting up to 15% of couples, can have varied causes ranging from natural ageing to the pathological development or function of the reproductive organs. One form of female infertility is premature ovarian insufficiency (POI), affecting up to 1 in 100 women and characterised by amenorrhoea and elevated FSH before the age of 40. POI can have a genetic basis, with over 50 causative genes identified. Non-obstructive azoospermia (NOA), a form of male infertility characterised by the absence of sperm in semen, has an incidence of 1% and is similarly heterogeneous. The genetic basis of male and female infertility is poorly understood with the majority of cases having no known cause. Here, we study a case of familial infertility including a proband with POI and her brother with NOA. We performed whole-exome sequencing (WES) and identified a homozygous STAG3 missense variant that segregated with infertility. STAG3 encodes a component of the meiosis cohesin complex required for sister chromatid separation. We report the first pathogenic homozygous missense variant in STAG3 and the first STAG3 variant associated with both male and female infertility. We also demonstrate limitations of WES for the analysis of homologous DNA sequences, with this variant being ambiguous or missed by independent WES protocols and its homozygosity only being established via long-range nested PCR.
Assuntos
Azoospermia/genética , Proteínas de Ciclo Celular/genética , Mutação de Sentido Incorreto , Insuficiência Ovariana Primária/genética , Adulto , Consanguinidade , Feminino , Homozigoto , Humanos , Infertilidade Feminina/genética , Infertilidade Masculina/genética , Masculino , Linhagem , IrmãosRESUMO
BACKGROUND: Cryptorchidism or failure of testicular descent is the most common genitourinary birth defect in males. While both the insulin-like peptide 3 (INSL3) and its receptor, relaxin family peptide receptor 2 (RXFP2), have been demonstrated to control testicular descent in mice, their link to human cryptorchidism is weak, with no clear cause-effect demonstrated. OBJECTIVE: To identify the genetic cause of a case of familial cryptorchidism. METHODS: We recruited a family in which four boys had isolated bilateral cryptorchidism. A fourth-degree consanguineous union in the family was reported. Whole exome sequencing was carried out for the four affected boys and their parents, and variants that segregated with the disorder and had a link to testis development/descent were analysed. Functional analysis of a RXFP2 variant in cell culture included receptor localisation, ligand binding and cyclic AMP (cAMP) pathway activation. RESULTS: Genomic analysis revealed a homozygous missense variant in the RXFP2 gene (c.1496G>A .p.Gly499Glu) in all four affected boys and heterozygous in both parents. No other variant with a link to testis biology was found. The RXFP2 variant is rare in genomic databases and predicted to be damaging. It has not been previously reported. Functional analysis demonstrated that the variant protein had poor cell surface expression and failed to bind INSL3 or respond to the ligand with cAMP signalling. CONCLUSION: This is the first reported genomic analysis of a family with multiple individuals affected with cryptorchidism. It demonstrates that recessive variants in the RXFP2 gene underlie familial cryptorchidism and solidifies the link between this gene and testicular descent in humans.
Assuntos
Criptorquidismo/genética , Genes Recessivos/genética , Mutação de Sentido Incorreto/genética , Receptores Acoplados a Proteínas G/genética , Linhagem Celular , Células HEK293 , Humanos , Masculino , Transdução de Sinais/genética , Testículo/patologiaRESUMO
BACKGROUND: Desert hedgehog (DHH) gene variants are known to cause 46,XY differences/disorders of sex development (DSD). We have identified six patients with 46,XY DSD with seven novel DHH gene variants. Many of these variants were classified as variants of uncertain significance due to their heterozygosity or associated milder phenotype. To assess variant pathogenicity and to refine the spectrum of DSDs associated with this gene, we have carried out the first reported functional testing of DHH gene variant activity. METHODS: A cell co-culture method was used to assess DHH variant induction of Hedgehog signalling in cultured Leydig cells. Protein expression and subcellular localisation were also assessed for DHH variants using western blot and immunofluorescence. RESULTS: Our co-culture method provided a robust read-out of DHH gene variant activity, which correlated closely with patient phenotype severity. While biallelic DHH variants from patients with gonadal dysgenesis showed significant loss of activity, variants found as heterozygous in patients with milder phenotypes had no loss of activity when tested with a wild type allele. Taking these functional results into account improved clinical interpretation. CONCLUSION: Our findings suggest heterozygous DHH gene variants are unlikely to cause DSD, reaffirming that DHH is an autosomal recessive cause of 46,XY gonadal dysgenesis. Functional characterisation of novel DHH variants improves variant interpretation, leading to greater confidence in patient reporting and clinical management.
Assuntos
Transtorno 46,XY do Desenvolvimento Sexual/diagnóstico , Transtorno 46,XY do Desenvolvimento Sexual/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Variação Genética , Proteínas Hedgehog/genética , Alelos , Células Cultivadas , Análise Mutacional de DNA , Expressão Gênica , Estudos de Associação Genética/métodos , Genótipo , Disgenesia Gonadal 46 XY/diagnóstico , Disgenesia Gonadal 46 XY/genética , Proteínas Hedgehog/metabolismo , Humanos , Células Intersticiais do Testículo/metabolismo , Masculino , Mutação , FenótipoRESUMO
Several recent reports have described a missense variant in the gene NR5A1 (c.274C>T; p.Arg92Trp) in a significant number of 46,XX ovotesticular or testicular disorders of sex development (DSDs) cases. The affected residue falls within the DNA-binding domain of the NR5A1 protein, however the exact mechanism by which it causes testicular development in 46,XX individuals remains unclear. We have screened a cohort of 26 patients with 46,XX (ovo)testicular DSD and identified three unrelated individuals with this NR5A1 variant (p.Arg92Trp), as well as one patient with a novel NR5A1 variant (c.779C>T; p.Ala260Val). We examined the functional effect of these changes, finding that while protein levels and localization were unaffected, variant NR5A1 proteins repress the WNT signaling pathway and have less ability to upregulate the anti-testis gene NR0B1. These findings highlight how NR5A1 variants impact ovarian differentiation across multiple pathways, resulting in a switch from ovarian to testis development in genetic females.
Assuntos
Transtornos 46, XX do Desenvolvimento Sexual/genética , Transtornos do Desenvolvimento Sexual/genética , Fator Esteroidogênico 1/genética , Testículo/patologia , Transtornos 46, XX do Desenvolvimento Sexual/patologia , Adolescente , Adulto , Pré-Escolar , Proteínas de Ligação a DNA/genética , Transtornos do Desenvolvimento Sexual/patologia , Feminino , Humanos , Lactente , Masculino , Mutação de Sentido Incorreto/genética , Linhagem , Fenótipo , Domínios Proteicos/genética , Testículo/crescimento & desenvolvimento , Via de Sinalização Wnt/genéticaRESUMO
Variants in the NR5A1 gene encoding SF1 have been described in a diverse spectrum of disorders of sex development (DSD). Recently, we reported the use of a targeted gene panel for DSD where we identified 15 individuals with a variant in NR5A1, nine of which are novel. Here, we examine the functional effect of these changes in relation to the patient phenotype. All novel variants tested had reduced trans-activational activity, while several had altered protein level, localization, or conformation. In addition, we found evidence of new roles for SF1 protein domains including a region within the ligand binding domain that appears to contribute to SF1 regulation of Müllerian development. There was little correlation between the severity of the phenotype and the nature of the NR5A1 variant. We report two familial cases of NR5A1 deficiency with evidence of variable expressivity; we also report on individuals with oligogenic inheritance. Finally, we found that the nature of the NR5A1 variant does not inform patient outcomes (including pubertal androgenization and malignancy risk). This study adds nine novel pathogenic NR5A1 variants to the pool of diagnostic variants. It highlights a greater need for understanding the complexity of SF1 function and the additional factors that contribute.
Assuntos
Transtornos do Desenvolvimento Sexual/diagnóstico , Transtornos do Desenvolvimento Sexual/genética , Estudos de Associação Genética , Variação Genética , Fenótipo , Fator Esteroidogênico 1/genética , Alelos , Sequência de Aminoácidos , Transtorno 46,XY do Desenvolvimento Sexual/diagnóstico , Transtorno 46,XY do Desenvolvimento Sexual/genética , Feminino , Estudos de Associação Genética/métodos , Genótipo , Humanos , Masculino , Modelos Anatômicos , Mutação , Conformação Proteica , Domínios Proteicos/genética , Sítios de Splice de RNA , Análise de Sequência de DNA , Fator Esteroidogênico 1/químicaRESUMO
BACKGROUND: Congenital hypogonadotrophic hypogonadism (CHH) and Kallmann syndrome (KS) are caused by disruption to the hypothalamic-pituitary-gonadal (H-P-G) axis. In particular, reduced production, secretion or action of gonadotrophin-releasing hormone (GnRH) is often responsible. Various genes, many of which play a role in the development and function of the GnRH neurons, have been implicated in these disorders. Clinically, CHH and KS are heterogeneous; however, in 46,XY patients, they can be characterised by under-virilisation phenotypes such as cryptorchidism and micropenis or delayed puberty. In rare cases, hypospadias may also be present. RESULTS: Here, we describe genetic mutational analysis of CHH genes in Indonesian 46,XY disorder of sex development patients with under-virilisation. We present 11 male patients with varying degrees of under-virilisation who have rare variants in known CHH genes. Interestingly, many of these patients had hypospadias. CONCLUSIONS: We postulate that variants in CHH genes, in particular PROKR2, PROK2, WDR11 and FGFR1 with CHD7, may contribute to under-virilisation phenotypes including hypospadias in Indonesia.
Assuntos
Hipogonadismo/congênito , Hipogonadismo/genética , Mutação , Adolescente , Criança , Pré-Escolar , Estudos de Coortes , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Hormônios Gastrointestinais/genética , Humanos , Hipogonadismo/patologia , Indonésia , Lactente , Masculino , Proteínas de Membrana/genética , Neuropeptídeos/genética , Proteínas Proto-Oncogênicas/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptores Acoplados a Proteínas G/genética , Receptores de Peptídeos/genéticaRESUMO
Disorders of Sex Development (DSDs) are a major paediatric concern and are estimated to occur in around 1.7% of all live births (Fausto-Sterling, Sexing the Body: Gender Politics and the Construction of Sexuality, Basic Books, New York, 2000). They are often caused by the breakdown in the complex genetic mechanisms that underlie gonadal development and differentiation. Having a genetic diagnosis can be important for patients with a DSD: it can increase acceptance of a disorder often surrounded by stigma, alter clinical management and it can assist in reproductive planning. While Massively Parallel Sequencing (MPS) is advancing the genetic diagnosis of rare Mendelian disorders, it is not yet clear which MPS assay is best suited for the clinical diagnosis of DSD patients and to what extent other established methods are still relevant. To complicate matters, DSDs represent a wide spectrum of disorders caused by an array of different genetic changes, many of which are yet unknown. Here we discuss the different genetic lesions that are known to contribute to different DSDs, and review the utility of a range of MPS approaches for diagnosing DSD patients. Birth Defects Research (Part C) 108:337-350, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Transtornos do Desenvolvimento Sexual/diagnóstico , Transtornos do Desenvolvimento Sexual/genética , Animais , Variações do Número de Cópias de DNA , Transtornos do Desenvolvimento Sexual/metabolismo , Estudos de Associação Genética , Predisposição Genética para Doença , Testes Genéticos , Genômica/métodos , Transtornos Gonadais/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/estatística & dados numéricos , Humanos , Desenvolvimento Sexual/genéticaRESUMO
In the female gonad, distinct signalling pathways activate ovarian differentiation while repressing the formation of testes. Human disorders of sex development (DSDs), such as 46,XX DSDs, can arise when this signalling is aberrant. Here we review the current understanding of the genetic mechanisms that control gonadal development, with particular emphasis on those that drive or inhibit ovarian differentiation. We discuss how disruption to these molecular pathways can lead to 46,XX disorders of ovarian development. Finally, we look at recently characterized novel genes and pathways that contribute and speculate how advances in technology will aid in further characterization of normal and disrupted human ovarian development.
Assuntos
Transtornos 46, XX do Desenvolvimento Sexual/genética , Animais , Gônadas/embriologia , HumanosRESUMO
BACKGROUND: Despite some advances in recent years, the genetic control of gonadal sex differentiation during embryogenesis is still not completely understood. To identify new candidate genes involved in ovary and testis development, RNA-seq was used to define the transcriptome of embryonic chicken gonads at the onset of sexual differentiation (day 6.0/stage 29). RESULTS: RNA-seq revealed more than 1000 genes that were transcribed in a sex-biased manner at this early stage of gonadal differentiation. Comparison with undifferentiated gonads revealed that sex biased expression was derived primarily from autosomal rather than sex-linked genes. Gene ontology and pathway analysis indicated that many of these genes encoded proteins involved in extracellular matrix function and cytoskeletal remodelling, as well as tubulogenesis. Several of these genes are novel candidate regulators of gonadal sex differentiation, based on sex-biased expression profiles that are altered following experimental sex reversal. We further characterised three female-biased (ovarian) genes; calpain-5 (CAPN5), G-protein coupled receptor 56 (GPR56), and FGFR3 (fibroblast growth factor receptor 3). Protein expression of these candidates in the developing ovaries suggests that they play an important role in this tissue. CONCLUSIONS: This study provides insight into the earliest steps of vertebrate gonad sex differentiation, and identifies novel candidate genes for ovarian and testicular development.
Assuntos
Estudos de Associação Genética , Gônadas/metabolismo , Diferenciação Sexual/genética , Animais , Embrião de Galinha , Análise por Conglomerados , Desenvolvimento Embrionário/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Análise de Sequência de RNA , TranscriptomaRESUMO
During development, secreted morphogens, such as Hedgehog (Hh), control cell fate and proliferation. Precise sensing of morphogen levels and dynamic cellular responses are required for morphogen-directed morphogenesis, yet the molecular mechanisms responsible are poorly understood. Several recent studies have suggested the involvement of a multi-protein Hh reception complex, and have hinted at an understated complexity in Hh sensing at the cell surface. We show here that the expression of the proteoglycan Dally in Hh-receiving cells in Drosophila is necessary for high but not low level pathway activity, independent of its requirement in Hh-producing cells. We demonstrate that Dally is necessary to sequester Hh at the cell surface and to promote Hh internalisation with its receptor. This internalisation depends on both the activity of the hydrolase Notum and the glycosyl-phosphatidyl-inositol (GPI) moiety of Dally, and indicates a departure from the role of the second glypican Dally-like in Hh signalling. Our data suggest that hydrolysis of the Dally-GPI by Notum provides a switch from low to high level signalling by promoting internalisation of the Hh-Patched ligand-receptor complex.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Proteínas Hedgehog/metabolismo , Glicoproteínas de Membrana/metabolismo , Morfogênese/fisiologia , Proteoglicanas/metabolismo , Transdução de Sinais/fisiologia , Animais , Animais Geneticamente Modificados , Western Blotting , Células Cultivadas , Processamento de Imagem Assistida por Computador , Microscopia de FluorescênciaRESUMO
In mammals, the primary role of anti-Müllerian hormone (AMH) during development is the regression of Müllerian ducts in males. These structures otherwise develop into fallopian tubes, oviducts, and upper vagina, as in females. This highly conserved function is retained in birds and is supported by the high levels of AMH expression in developing testes. In mammals, AMH expression is controlled partly by the transcription factor, SOX9. However, in the chicken, AMH mRNA expression precedes that of SOX9 , leading to the view that AMH may lie upstream of SOX9 and play a more central role in avian testicular development. To help define the role of AMH in chicken gonad development, we suppressed AMH expression in chicken embryos using RNA interference. In males, AMH knockdown did not affect the expression of key testis pathway genes, and testis cords developed normally. However, a reduction in the size of the mesonephros and gonads was observed, a phenotype that was evident in both sexes. This growth defect occurred as a result of the reduced proliferative capacity of the cells of these tissues, and male gonads also had a significant reduction in germ cell numbers. These data suggest that although AMH does not directly contribute to testicular or ovarian differentiation, it is required in a sex-independent manner for proper cell proliferation and urogenital system growth.
Assuntos
Hormônio Antimülleriano/genética , Ovário/embriologia , Diferenciação Sexual/genética , Testículo/embriologia , Sistema Urogenital/embriologia , Animais , Hormônio Antimülleriano/metabolismo , Embrião de Galinha , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Ovário/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Testículo/metabolismo , Sistema Urogenital/metabolismoRESUMO
Anti-Müllerian hormone (AMH) signaling is required for proper development of the urogenital system in vertebrates. In male mammals, AMH is responsible for regressing the Müllerian ducts, which otherwise develop into the fallopian tubes, oviducts, and upper vagina of the female reproductive tract. This role is highly conserved across higher vertebrates. However, AMH is required for testis development in fish species that lack Müllerian ducts, implying that AMH signaling has broader roles in other vertebrates. AMH signals through two serine/threonine kinase receptors. The primary AMH receptor, AMH receptor type-II (AMHR2), recruits the type I receptor, which transduces the signal intracellularly. To enhance our understanding of AMH signaling and the potential role of AMH in gonadal sex differentiation, we cloned chicken AMHR2 cDNA and examined its expression profile during gonadal sex differentiation. AMHR2 is expressed in the gonads and Müllerian ducts of both sexes but is more strongly expressed in males after the onset of gonadal sex differentiation. In the testes, the AMHR2 protein colocalizes with AMH, within Sertoli cells of the testis cords. AMHR2 protein expression is up-regulated in female embryos treated with the estrogen synthesis inhibitor fadrozole. Conversely, knockdown of the key testis gene DMRT1 leads to disruption of AMHR2 expression in the developing seminiferous cords of males. These results indicate that AMHR2 is developmentally regulated during testicular differentiation in the chicken embryo. AMH signaling may be important for gonadal differentiation in addition to Müllerian duct regression in birds.
Assuntos
Galinhas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Gônadas/metabolismo , Receptores de Peptídeos/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Diferenciação Sexual/fisiologia , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Embrião de Galinha , Clonagem Molecular , Feminino , Hibridização In Situ/veterinária , Masculino , Dados de Sequência Molecular , Filogenia , RNA/química , RNA/genética , Receptores de Peptídeos/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Alinhamento de Sequência , Análise de Sequência de DNA , Fatores de Transcrição/metabolismoRESUMO
: Differences/disorders of sex development can be caused by disruptions to the molecular and cellular mechanisms that control development and sex determination of the reproductive organs with 1:100 live births affected. Multiple genes are associated with 46, XY differences/disorders of sex development that can cause varying clinical phenotypes. An accurate genetic diagnosis is essential to guide clinical care for individuals with 46, XY differences/disorders of sex development and can contribute to family planning. The use of genomics in differences/disorders of sex development has grown, with several advances employed in genetic diagnosis; however, diagnostic rates have stagnated at less than 50% for these conditions. This review will discuss 46, XY differences/disorders of sex development, its molecular causes, and the genomic technologies currently utilized for diagnosis with focus on reports from the last 5 years. We also touch on the challenges in diagnosing 46, XY differences/disorders of sex development and discuss new and future technologies that promise to improved diagnostic rates for these difficult conditions.
RESUMO
BACKGROUND: The Cut Homeobox 1 (CUX1) gene has been implicated in a number of developmental processes and has recently emerged as an important cause of developmental delay and impaired intellectual development. Individuals with variants in CUX1 have been described with a variety of co-morbidities including variations in sex development (VSD) although these features have not been closely documented. CASE PRESENTATION: The proband is a 14-year-old male who presented with congenital complex hypospadias, neurodevelopmental differences, and subtle dysmorphism. A family history of neurodevelopmental differences and VSD was noted. Microarray testing and whole exome sequencing found the 46,XY proband had a large heterozygous in-frame deletion of exons 4-10 of the CUX1 gene. CONCLUSIONS: Our review of the literature has revealed that variants in CUX1 are associated with a range of VSD and suggest this gene should be considered in cases where a VSD is noted at birth, especially if there is a familial history of VSD and/or neurodevelopmental differences. Further work is required to fully investigate the role and regulation of CUX1 in sex development.