scholarly journals Establishment of a Novel Human Fetal Adrenal Culture Model that Supports de Novo and Manipulated Steroidogenesis

2020 ◽  
Author(s):  
Cecilie Melau ◽  
John E Nielsen ◽  
Signe Perlman ◽  
Lene Lundvall ◽  
Lea Langhoff Thuesen ◽  
...  
Keyword(s):  
De Novo ◽  
Culture Media ◽  
Ex Vivo ◽  
Disorders Of Sex Development ◽  
Endocrine Function ◽  
Specific Response ◽  
Adrenal Tissue ◽  
Sex Development ◽  
Adrenal Steroidogenesis ◽  
Novel Approach

Abstract Context Disorders affecting adrenal steroidogenesis promote an imbalance in the normally tightly controlled secretion of mineralocorticoids, glucocorticoids, and androgens. This may lead to differences/disorders of sex development in the fetus, as seen in virilized girls with congenital adrenal hyperplasia (CAH). Despite the important endocrine function of human fetal adrenals, neither normal nor dysregulated adrenal steroidogenesis is understood in detail. Objective Due to significant differences in adrenal steroidogenesis between human and model species (except higher primates), we aimed to establish a human fetal adrenal model that enables examination of both de novo and manipulated adrenal steroidogenesis. Design and Setting Human adrenal tissue from 54 1st trimester fetuses were cultured ex vivo as intact tissue fragments for 7 or 14 days. Main Outcome Measures Model validation included examination of postculture tissue morphology, viability, apoptosis, and quantification of steroid hormones secreted to the culture media measured by liquid chromatography-tandem mass spectrometry. Results The culture approach maintained cell viability, preserved cell populations of all fetal adrenal zones, and recapitulated de novo adrenal steroidogenesis based on continued secretion of steroidogenic intermediates, glucocorticoids, and androgens. Adrenocorticotropic hormone and ketoconazole treatment of ex vivo cultured human fetal adrenal tissue resulted in the stimulation of steroidogenesis and inhibition of androgen secretion, respectively, demonstrating a treatment-specific response. Conclusions Together, these data indicate that ex vivo culture of human fetal adrenal tissue constitutes a novel approach to investigate local effects of pharmaceutical exposures or emerging therapeutic options targeting imbalanced steroidogenesis in adrenal disorders, including CAH.

MYRF haploinsufficiency causes 46,XY and 46,XX disorders of sex development: bioinformatics consideration

2019 ◽  
Vol 28 (14) ◽  
pp. 2319-2329 ◽  
Author(s):  
Kohei Hamanaka ◽  
Atsushi Takata ◽  
Yuri Uchiyama ◽  
Satoko Miyatake ◽  
Noriko Miyake ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Target Genes ◽  
De Novo ◽  
Clinical Symptoms ◽  
Disorders Of Sex Development ◽  
Sequencing Data ◽  
Causative Gene ◽  
Sex Development ◽  
And Migration ◽  
Proliferation And Migration

AbstractDisorders of sex development (DSDs) are defined as congenital conditions in which chromosomal, gonadal or anatomical sex is atypical. In many DSD cases, genetic causes remain to be elucidated. Here, we performed a case–control exome sequencing study comparing gene-based burdens of rare damaging variants between 26 DSD cases and 2625 controls. We found exome-wide significant enrichment of rare heterozygous truncating variants in the MYRF gene encoding myelin regulatory factor, a transcription factor essential for oligodendrocyte development. All three variants occurred de novo. We identified an additional 46,XY DSD case of a de novo damaging missense variant in an independent cohort. The clinical symptoms included hypoplasia of Müllerian derivatives and ovaries in 46,XX DSD patients, defective development of Sertoli and Leydig cells in 46,XY DSD patients and congenital diaphragmatic hernia in one 46,XY DSD patient. As all of these cells and tissues are or partly consist of coelomic epithelium (CE)-derived cells (CEDC) and CEDC developed from CE via proliferaiton and migration, MYRF might be related to these processes. Consistent with this hypothesis, single-cell RNA sequencing of foetal gonads revealed high expression of MYRF in CE and CEDC. Reanalysis of public chromatin immunoprecipitation sequencing data for rat Myrf showed that genes regulating proliferation and migration were enriched among putative target genes of Myrf. These results suggested that MYRF is a novel causative gene of 46,XY and 46,XX DSD and MYRF is a transcription factor regulating CD and/or CEDC proliferation and migration, which is essential for development of multiple organs.

scholarly journals FGF9 is a downstream target of SRY and sufficient to determine male sex fate in ex vivo XX gonad culture

2020 ◽  
Vol 103 (6) ◽  
pp. 1300-1313
Author(s):  
Yi-Han Li ◽  
Tsung-Ming Chen ◽  
Bu-Miin Huang ◽  
Shang-Hsun Yang ◽  
Chia-Ching Wu ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Fate ◽  
Ex Vivo ◽  
Disorders Of Sex Development ◽  
Sex Reversal ◽  
Vital Role ◽  
Loss Of Function ◽  
Downstream Target ◽  
Sex Development ◽  
Male Sex

Abstract Fibroblast growth factor 9 (FGF9) is an autocrine/paracrine growth factor that plays critical roles in embryonic and organ developments and is involved in diverse physiological events. Loss of function of FGF9 exhibits male-to-female sex reversal in the transgenic mouse model and gain of FGF9 copy number was found in human 46, XX sex reversal patient with disorders of sex development. These results suggested that FGF9 plays a vital role in male sex development. Nevertheless, how FGF9/Fgf9 expression is regulated during testis determination remains unclear. In this study, we demonstrated that human and mouse SRY bind to −833 to −821 of human FGF9 and −1010 to −998 of mouse Fgf9, respectively, and control FGF9/Fgf9 mRNA expression. Interestingly, we showed that mouse SRY cooperates with SF1 to regulate Fgf9 expression, whereas human SRY-mediated FGF9 expression is SF1 independent. Furthermore, using an ex vivo gonadal culture system, we showed that FGF9 expression is sufficient to switch cell fate from female to male sex development in 12–16 tail somite XX mouse gonads. Taken together, our findings provide evidence to support the SRY-dependent, fate-determining role of FGF9 in male sex development.

scholarly journals OR15-07 Novel Genes Involved in Sex Differentiation Identified by Whole-Exome Sequencing in a Cohort of Children with Disorders of Sex Development

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Yardena Tenenbaum Rakover ◽  
Osnat Admoni ◽  
Ghadir Elias Assad ◽  
Shira London ◽  
Marie Noufi Barhoum ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
De Novo ◽  
Disorders Of Sex Development ◽  
External Genitalia ◽  
De Novo Mutation ◽  
Novel Genes ◽  
Sex Development ◽  
First Case ◽  
Whole Exome

Abstract Background: Disorders of sex development (DSD) are classified as a congenital discrepancy between external genitalia, gonadal and chromosomal sex. Despite extensive laboratory and imaging investigations, the etiology of DSD is unknown in more than 50% of patients and the diagnosis is often delayed to the second decade of life. Our objective was to evaluate the etiology of DSD by whole-exome sequencing (WES) in children in whom hormonal and candidate gene approaches had not identified the etiology. Methods: Nine patients diagnosed with DSD (eight 46,XY and one 46,XX) were enrolled. Patients underwent hormonal evaluation, including ACTH, GnRH and hCG tests. Candidate genes were sequenced in accordance with the hormonal results. WES was performed for the probands and their parents. Results: The eight 46,XY patients presented with micropenis, cryptorchidism and hypospadias at birth and the 46,XX patient with fusion of the labia majora. In six of the nine patients (66%), a pathogenic mutation was identified by WES that explained the phenotype: four known DSD-causing genes—POR, CHD7, HSD17B3 and WT1—and two novel genes—BMP4 and RFXP2. In three patients, variants of unknown significance were found. An 11-y-old boy had a novel de-novo mutation in BMP4. In humans, mutations in this gene, encoding bone morphogenetic protein 4, are associated with autosomal dominant microphthalmia. BMP4 is expressed in the urethral epithelium and has a role in the development of external genitalia and the pituitary. This is the first report of a BMP4 mutation in a child with DSD. A 12-y-old boy had a mutation in RFXP2, encoding insulin-like 3 hormone receptor, which has been previously reported in adult males with cryptorchidism. This is the first case of an RFXP2 mutation in a child with DSD. Conclusions: WES has a crucial role in early diagnosis of the etiology of DSD, making extensive endocrine testing unnecessary, and has important implications for sex of rearing decisions.

scholarly journals A de novo derivative Y chromosome (partial Yq deletion and partial duplication of Yp and Yq) in a female with disorders of sex development

2018 ◽  
Vol 6 (9) ◽  
pp. 1671-1676
Author(s):  
Qing-Song Liu ◽  
Xing-Chun Zhu ◽  
Qiang Ma ◽  
Cheng He ◽  
Jian-Lan Shao
Keyword(s):  
Y Chromosome ◽  
De Novo ◽  
Disorders Of Sex Development ◽  
Partial Duplication ◽  
Sex Development

scholarly journals Intragenic Duplication of DMRT1 in a SRY-Negative Boy with 46,XX Disorder of Sex Development

2021 ◽  
Author(s):  
Veronica Bertini ◽  
Fulvia Baldinotti ◽  
Nina Tyutyusheva ◽  
Camillo Rosano ◽  
Cinzia Cosini ◽  
...  
Keyword(s):  
De Novo ◽  
Sex Differentiation ◽  
Disorders Of Sex Development ◽  
Comparative Genomic ◽  
Normal Male ◽  
Testicular Development ◽  
Targeted Next Generation Sequencing ◽  
Sex Development ◽  
Intragenic Duplication ◽  
Xx Males

Background. 46,XX disorders of sex development are rare. Approximately, 90% of XX males are SRY-positive, while testicular development in the absence of SRY takes place in a minority. Methods: A boy with 46,XX karyotype (SRY-negative; absence of SOX9 duplications) was investigated by targeted Next Generation Sequencing (NGS), Multiplex ligation-dependent probe amplification (MLPA), and Comparative Genomic Hybridization array (CGH-array). Results: The boy had normal male phenotype and normal prepubertal values of testicular hormones. He presented a heterozygous duplication of 49.626 bp, encompassing exons 2 and 3 of DMRT1. The result was arr[GRCh37] 9p24.3(845893_895518)x3. Since both breakpoints are harbored in the intronic regions, the duplication does not stop or shift the coding frame. Additional known pathogenic or uncertain variants in pro-testis gene cascade were not identified. Conclusions: This study report a boy with 46,XX testicular disorder of sex differentiation, showing a de novo partial intragenic duplication of DMRT1. This intragenic duplication may result in a gain of function, acting as primary pro-testis gene (or anti-ovary gene) in a 46,XX human foetus and permitting normal pre-pubertal endocrine testis function.

Chromosomal Disorders of Sex Development

2018 ◽  
Author(s):  
R. J McKinlay Gardner ◽  
David J Amor
Keyword(s):  
Sexual Development ◽  
De Novo ◽  
Chromosome Abnormality ◽  
Disorders Of Sex Development ◽  
External Genitalia ◽  
Sequential Process ◽  
Chromosomal Disorders ◽  
Sex Development ◽  
Development Proceeds

Chromosomal sex is, for the most part, congruently XX female and XY male. The XX and XY embryo are built on a fundamentally similar outline plan, and only as development proceeds do certain modifications evolve. If at any point in this sequential process some genetic instruction is faulty, inappropriate, or cannot be acted on, the direction of anatomical sexual development may proceed imperfectly or completely incongruently. This chapter reviews the conditions of ambiguous/incomplete/indeterminate development of the internal and external genitalia, where the basis of this is a chromosome abnormality, usually of the X or the Y chromosome. The key role of the SRY male-determining gene in a number of these conditions is noted. The de novo or familial origin of these disorders is discussed, with particular reference to possible risks of recurrence.

scholarly journals The evolving role of whole-exome sequencing in the management of disorders of sex development

2021 ◽  
Author(s):  
Yardena Tenenbaum Rakover ◽  
Osnat Admoni ◽  
Gadir Elias-Assad ◽  
Shira London ◽  
Marie Noufi Barhoum ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
De Novo ◽  
Disorders Of Sex Development ◽  
External Genitalia ◽  
Sex Development ◽  
Pathogenic Variants ◽  
Extensive Evaluation ◽  
Whole Exome

Objective: Disorders of sex development (DSD) are defined as congenital conditions in which development of chromosomal, gonadal and anatomical sex is atypical. Despite wide laboratory and imaging investigations, the etiology of DSD is unknown in over 50% of patients. Methods: We evaluated the etiology of DSD by whole-exome sequencing (WES) at a mean age of 10 years in nine patients for whom extensive evaluation, including hormonal, imaging and candidate gene approaches, had not identified an etiology. Results: The eight 46,XY patients presented with micropenis, cryptorchidism and hypospadias at birth and the 46,XX patient presented with labia majora fusion. In seven patients (78%), pathogenic variants were identified for RXFP2, HSD17B3, WT1, BMP4, POR, CHD7 and SIN3A. In two patients, no causative variants were found. Mutations in three genes were reported previously with different phenotypes: an 11-year-old boy with a novel de novo variant in BMP4; such variants are mainly associated with microphthalmia and in few cases with external genitalia anomalies in males, supporting the role of BMP4 in the development of male external genitalia; a 12-year-old boy with a known pathogenic variant in RXFP2, encoding insulin-like 3 hormone receptor, and previously reported in adult men with cryptorchidism; an 8-year-old boy with syndromic DSD had a de novo deletion in SIN3A. Conclusions: Our findings of molecular etiologies for DSD in 78% of our patients indicate a major role for WES in early DSD diagnosis and management, and highlights the importance of rapid molecular diagnosis in early infancy for sex of rearing decisions.

scholarly journals Heterozygous Missense Mutations in Steroidogenic Factor 1 (SF1/Ad4BP, NR5A1) Are Associated with 46,XY Disorders of Sex Development with Normal Adrenal Function

2007 ◽  
Vol 92 (3) ◽  
pp. 991-999 ◽  
Author(s):  
Lin Lin ◽  
Pascal Philibert ◽  
Bruno Ferraz-de-Souza ◽  
Daniel Kelberman ◽  
Tessa Homfray ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
De Novo ◽  
Mutational Analysis ◽  
Case Reports ◽  
Disorders Of Sex Development ◽  
Adrenal Function ◽  
Testicular Function ◽  
Missense Mutations ◽  
Steroidogenic Factor 1 ◽  
Sex Development

Abstract Context: Steroidogenic factor 1 (SF1/AdBP4/FTZF1, NR5A1) is a nuclear receptor transcription factor that plays a key role in regulating adrenal and gonadal development, steroidogenesis, and reproduction. Targeted deletion of Nr5a1 (Sf1) in the mouse results in adrenal and gonadal agenesis, XY sex-reversal, and persistent Müllerian structures in males. Consistent with the murine phenotype, human mutations in SF1 were described initially in two 46,XY individuals with female external genitalia, Müllerian structures (uterus), and primary adrenal failure. Objective: Given recent case reports of haploinsufficiency of SF1 affecting testicular function in humans, we aimed to identify SF1 mutations in a cohort of individuals with a phenotypic spectrum of 46,XY gonadal dysgenesis/impaired androgenization (now termed 46,XY disorders of sex development) with normal adrenal function. Methods and Patients: The study included mutational analysis of NR5A1 in 30 individuals with 46,XY disorders of sex development, followed by functional studies of SF1 activity. Results: Heterozygous missense mutations in NR5A1 were found in four individuals (four of 30, 13%) with this phenotype. These mutations (V15M, M78I, G91S, L437Q) were shown to impair transcriptional activation through abnormal DNA binding (V15M, M78I, G91S), altered subnuclear localization (V15M, M78I), or disruption of the putative ligand-binding pocket (L437Q). Two mutations appeared to be de novo or germline changes. The other two mutations appeared to be inherited in a sex-limited dominant manner because the mother is heterozygous for the change. Conclusions: These studies demonstrate that SF1 mutations are more frequent than previously suspected causes of impaired fetal and postnatal testicular function in 46,XY individuals.

scholarly journals The influence of the gut microbiome on BCG-induced trained immunity

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Martin Stražar ◽  
Vera P. Mourits ◽  
Valerie A. C. M. Koeken ◽  
L. Charlotte J. de Bree ◽  
Simone J. C. F. M. Moorlag ◽  
...  
Keyword(s):  
Immune Responses ◽  
Gut Microbiome ◽  
De Novo ◽  
Ex Vivo ◽  
Specific Response ◽  
Dependent Manner ◽  
De Novo Genome Assembly ◽  
Bcg Vaccination ◽  
Vaccine Responses ◽  
Phenylalanine Metabolism

Abstract Background The bacillus Calmette-Guérin (BCG) vaccine protects against tuberculosis and heterologous infections but elicits high inter-individual variation in specific and nonspecific, or trained, immune responses. While the gut microbiome is increasingly recognized as an important modulator of vaccine responses and immunity in general, its potential role in BCG-induced protection is largely unknown. Results Stool and blood were collected from 321 healthy adults before BCG vaccination, followed by blood sampling after 2 weeks and 3 months. Metagenomics based on de novo genome assembly reveals 43 immunomodulatory taxa. The nonspecific, trained immune response is detected by altered production of cytokines IL-6, IL-1β, and TNF-α upon ex vivo blood restimulation with Staphylococcus aureus and negatively correlates with abundance of Roseburia. The specific response, measured by IFN-γ production upon Mycobacterium tuberculosis stimulation, is associated positively with Ruminococcus and Eggerthella lenta. The identified immunomodulatory taxa also have the strongest effects on circulating metabolites, with Roseburia affecting phenylalanine metabolism. This is corroborated by abundances of relevant enzymes, suggesting alternate phenylalanine metabolism modules are activated in a Roseburia species-dependent manner. Conclusions Variability in cytokine production after BCG vaccination is associated with the abundance of microbial genomes, which in turn affect or produce metabolites in circulation. Roseburia is found to alter both trained immune responses and phenylalanine metabolism, revealing microbes and microbial products that may alter BCG-induced immunity. Together, our findings contribute to the understanding of specific and trained immune responses after BCG vaccination.

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