Genetic reanalysis of patients with a difference of sex development carrying the NR5A1/SF-1 variant p.Gly146Ala has discovered other likely disease-causing variations

Sexual differentiation; Genital anatomy; PhenotypesDiferenciación sexual; Anatomía de los genitales; FenotiposDiferenciació sexual; Anatomia dels genitals; FenotipsNR5A1/SF-1 (Steroidogenic factor-1) variants may cause mild to severe differences of sex development (DSD) or may be found in healthy carriers. The NR5A1/SF-1 c.437G>C/p.Gly146Ala variant is common in individuals with a DSD and has been suggested to act as a susceptibility factor for adrenal disease or cryptorchidism. Since the allele frequency is high in the general population, and the functional testing of the p.Gly146Ala variant revealed inconclusive results, the disease-causing effect of this variant has been questioned. However, a role as a disease modifier is still possible given that oligogenic inheritance has been described in patients with NR5A1/SF-1 variants. Therefore, we performed next generation sequencing (NGS) in 13 DSD individuals harboring the NR5A1/SF-1 p.Gly146Ala variant to search for other DSD-causing variants and clarify the function of this variant for the phenotype of the carriers. Panel and whole-exome sequencing was performed, and data were analyzed with a filtering algorithm for detecting variants in NR5A1- and DSD-related genes. The phenotype of the studied individuals ranged from scrotal hypospadias and ambiguous genitalia in 46,XY DSD to opposite sex in both 46,XY and 46,XX. In nine subjects we identified either a clearly pathogenic DSD gene variant (e.g. in AR) or one to four potentially deleterious variants that likely explain the observed phenotype alone (e.g. in FGFR3, CHD7). Our study shows that most individuals carrying the NR5A1/SF-1 p.Gly146Ala variant, harbor at least one other deleterious gene variant which can explain the DSD phenotype. This finding confirms that the NR5A1/SF-1 p.Gly146Ala variant may not contribute to the pathogenesis of DSD and qualifies as a benign polymorphism. Thus, individuals, in whom the NR5A1/SF-1 p.Gly146Ala gene variant has been identified as the underlying genetic cause for their DSD in the past, should be re-evaluated with a NGS method to reveal the real genetic diagnosis.: This work has been supported by the University of the Basque Country (UPV-EHU) (Spain) (IT1739-22) and by the Swiss National Science Foundation (320030-197725). IM is supported by a Postdoctoral Fellowship Grant from the Education Department of Basque Government (Spain). JA is supported by ASONMEC (Asociacion de Oncologia Medica del Hospital de Cruces) (Spain). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Novel variant in HHAT as a cause of different sex development with partial gonadal dysgenesis associated with microcephaly, eye defects, and distal phalangeal hypoplasia of both thumbs: Case report

Different sexual development; Minigene studiesDesarrollo sexual diferente; Estudios de minigenesDesenvolupament sexual diferent; Estudis minigènicsThe palmitoylation of the Hedgehog (Hh) family of morphogens, named sonic hedgehog (SHH), desert hedgehog (DHH), and Indian hedgehog (IHH), is crucial for effective short- and long-range signaling. The hedgehog acyltransferase (HHAT) attaches the palmitate molecule to the Hh; therefore, variants in HHAT cause a broad spectrum of phenotypes. A missense HHAT novel variant c.1001T>A/p.(Met334Lys) was described in a patient first referred for a 46,XY different sexual development with partial gonadal dysgenesis but with microcephaly, eye defects, and distal phalangeal hypoplasia of both thumbs. The in silico analysis of the variant predicted an affectation of the nearest splicing site. Thus, in vitro minigene studies were carried out, which demonstrated that the variant does not affect the splicing. Subsequent protein in silico studies supported the pathogenicity of the variant, and, in conclusion, this was considered the cause of the patient’s phenotype.This study was partly supported by a grant from the Fondo de Investigación Sanitaria (PI15/01647 [to MF-C and SB-S])

CIBERER : Spanish national network for research on rare diseases: A highly productive collaborative initiative

Altres ajuts: Instituto de Salud Carlos III (ISCIII); Ministerio de Ciencia e Innovación.CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research

Inestabilitat cromosòmica transgeneracional i radioprotecció en rata

La radioteràpia és un dels tractaments indicats contra el càncer. En el cas de les dones, l'exposició a la radiació ionitzant (RI) pot produir una fallada en la funció ovàrica. Per tant, l'estudi exhaustiu dels efectes dels raigs X en les gònades femenines, i, més concretament, en les seves cèl·lules germinals, es fa necessari; així com la cerca d'un agent radioprotector, per tal de disminuir-ne aquests efectes secundaris. Aquesta tesi doctoral apropa la recerca bàsica a la clínica i els seus quatre objectius principals es centren en determinar l'existència dels efectes de la RI (raigs X), de mutàgens químics (afidicolina) i de possibles radioprotectors (triptorelina); així com la determinació de regions genòmicament inestables en l'espècie Rattus norvegicus. La línia germinal de rates femella ha estat la base d'aquest estudi. No obstant, les cèl·lules objecte d'anàlisi han estat les cèl·lules somàtiques (fibroblasts fetals) descendents dels oòcits I fecundats de les femelles adultes dels diferents grups de tractament. D'aquesta manera, s'ha pogut estudiar la inestabilitat cromosòmica radioinduïda (RICI) transgeneracional amb diferents paràmetres citogenètics. Paral·lelament, s'ha realitzat un estudi reproductiu analitzant directament les femelles tractades. S'ha demostrat que hi ha RICI transgeneracional. Els efectes de l'exposició a la RI d'oòcits I de rates femelles adultes es transmeten a la seva descendència. Aquesta RICI es presenta amb administració aguda i fraccionada de la dosi, i amb resultats semblants. Pel que fa a l'estudi reproductiu, no s'observa un efecte de la radiació amb els nostres paràmetres. Els fibroblasts fetals de l'espècie R. norvegicus presenten inestabilitat cromosòmica espontània. Aquesta s'observa tant pels paràmetres quantitatius com pels qualitatius i es manté en tots els experiments que s'han fet. L'administració del mutagen químic afidicolina als cultius de fibroblasts fetals de rata provoca dany citogenètic. Aquests efectes directes són clarament superiors quan el mutagen químic s'aplica a cèl·lules amb RICI transgeneracional indicant que aquesta en potencia els efectes. L'anàleg agonista de l'hormona GnRH estudiat, la triptorelina, confereix radioprotecció als oòcits I dels fol·licles primordials de les femelles adultes irradiades. El grau de radioprotecció d'aquest fàrmac depèn del paràmetre analitzat, de la dosi i del tipus d'administració de la radiació ionitzant. En paràmetres citogenètics, la radioprotecció d'aquest anàleg és molt més pronunciada a dosi aguda (fins a un 44 %). Traslladant els nostres resultat a l'espècie humana, els nostres resultats en rata indicarien que els ovaris prepuberals i els ovaris de pacients adultes tractades amb aquest anàleg agonista de la GnRHa són més radioresistents que els ovaris de pacients adultes no tractades amb l'anàleg, confirmant estudis clínics i histològics anteriors. La triptorelina també té un efecte protector enfront de la inestabilitat cromosòmica espontània, que es traduiria en una millora de la funció gonadal. Aquesta és evident quant als paràmetres citogenètics estudiats, i s'observa com a tendència pel que fa a les condicions reproductives de les femelles. En relació a l'estudi de regions inestables, s'han identificat disset seqüències telomèriques intersticials classificades com a freqüents i molt freqüents i s'han caracteritzat set llocs fràgils induïts per l'afidicolina en fibroblasts fetals d'aquesta espècie. Ambdós tipus de seqüències estan implicats en processos evolutius i en esdeveniments d'inestabilitat cromosòmica de l'espècie. Per acabar, s'ha determinat que les bandes cromosòmiques en rata no es trenquen a l'atzar, sinó que hi ha regions que pateixen més alteracions cromosòmiques que altres i que, per tant, són més inestables. Tenint en compte els efectes transgeneracionals de la RI, els efectes d'un mutagen químic, la inestabilitat cromosòmica espontània, els llocs fràgils i les seqüències telomèriques intersticials, les zones més inestables del cariotip de rata es localitzen en els cromosomes RNO1, RNO2 i RNO3. Les bandes cromosòmiques RNO1q22, RNO1q43, RNO2q32 i RNO3p12 són les més inestables del cariotip d'aquesta espècie.Radiotherapy is one of the indicated treatments against cancer. In women, exposure to ionising radiation (IR) can induce a failure in the ovarian function. Therefore, there is a need for an exhaustive analysis of the X-ray effects in the female gonads and to search for a radio protector agent, so as to decrease these secondary effects. This Ph.D. thesis attempts to infer conclusions from fundamental research and to apply this in a clinical capacity. Its four prime objectives are focused on the existence of effects of IR (X rays), of chemical mutagens (aphidicolin) and of possible radio protectors (triptorelin); and the determination of unstable genomic regions in the species Rattus norvegicus. The basis of this study was the germinal line of female rats. However, the objects of analysis were the somatic cells (foetal fibroblasts) originating from the oocytes I of adult females from different treatment groups. In this way, transgenerational radiation-induced chromosomal instability (RICI) was studied with different cytogenetical parameters. At the same time, a reproductive study was performed directly on the treated females. The existence of transgenerational RICI was demonstrated. Effects of the IR exposure of oocytes I from adult female rats are transmitted to their offspring. This RICI is present in acute and fractioned dosage administration, with similar results. Relating to the reproductive study, no radiation effect was seen with our parameters. Foetal fibroblasts of R. norvegicus present spontaneous chromosomal instability. It is observed with both the quantitative and qualitative parameters and it is maintained in all the experiments performed. Chemical mutagen aphidicolin administration to the foetal rat fibroblasts cultures causes cytogenetical damage. These direct effects are clearly superior when the chemical mutagen is applied to cells with transgenerational RICI, which indicates that RICI enhances its effects. The GnRH hormone agonist studied, triptorelin, confers radioprotection to the oocytes I from the primordial follicles of adult irradiated females. The degree of radioprotection of this drug depends on the analysed parameter, and on the dose and type of administration of ionising radiation. Relating to cytogenetical parameters, radioprotection of this analogue is much more pronounced at an acute dose (up to 44%). If we relate our results to the human species, they would indicate that prepubertal ovaries and those from adult patients treated with this GnRH agonist are more radioresistant than those from non-treated adult patients. This would confirm former histological and clinical studies. Triptorelin also affords protection from spontaneous chromosomal instability that would be translated into an improved gonadal function. This effect is evident relating to the cytogenetical parameters and it is observed as a tendency with respect to the reproductive conditions of the females. Concerning to the study of unstable regions, seventeen interstitial telomeric-like sequences, classified as frequent and very frequent, and seven aphidicolin-induced fragile sites in rat foetal fibroblasts were detected. Both types of sequences are involved in evolutive processes and in chromosomal instability events of this species. To finish, it was determined that chromosomal bands in rats do not break at random. There are chromosomal regions more unstable than others, as they suffer more chromosomal aberrations than others. The more unstable zones of the rat karyotype are located in chromosomes RNO1, RNO2 and RNO3, taking into account the transgenerational effects of IR, the effects of a chemical mutagen, the spontaneous chromosomal instability, fragile sites and interstitial telomeric-like sequences. Furthermore the more unstable chromosomal bands of this species are RNO1q22, RNO1q43, RNO2q32 and RNO3p12

Ovaris més resistents a la radioteràpia

L'eliminació de cèl·lules tumorals en el nostre organisme es basa en l'acció localitzada de radiacions ionitzants (raigs X, radioteràpia), una tecnologia sanitària efectiva, però no exempta d'inconvenients. D'entre ells, destaca la interrupció de la capacitat reproductiva de les dones, però sobretot les que ja menstruen. Sembla ser que els ovaris prepuberals de les noies resisteixen millor front les radiacions ionitzants que els de dones adultes. A partir d'aquest fet, els investigadors estudien la possibilitat d'aplicar fàrmacs, com ara la triptorelina, que actua com l'hormona alliberadora de gonadotrofines (GnRH), essencial per al correcte funcionament dels ovaris. De moment, assajos amb rates femella animen a continuar per aquest camí.La eliminación de células tumorales en nuestro organismo se basa en la acción localizada de radiaciones ionizantes (rayos X, radioterapia), una tecnología sanitaria efectiva, pero no exenta de inconvenientes. Entre ellos, destaca la interrupción de la capacidad reproductiva de las mujeres, pero sobre todo en aquéllas que ya menstrúan. Parece ser que los ovarios prepuberales de las chicas resisten mejor frente a las radiaciones ionizantes que los de mujeres adultas. A partir este hecho, los investigadores estudian la posibilidad de aplicar fármacos, como la Triptorelina, que actúa como la hormona liberadora de gonadotropinas (GnRH), esencial para el correcto funcionamiento de los ovarios. De momento, los ensayos con ratas hembra animan a continuar por este camino

A novel CYP17A1 deletion causes a functional knockout of the steroid enzyme 17-hydroxylase and 17,20-lyase in a Turkish family and illustrates the precise role of the CYP17A1 gene

A novel homozygous long-range deletion of the CYP17A1 gene abolished protein expression and caused the severest form of 17-hydroxylase deficiency in one kindred of a Turkish family. The affected subjects presented with 46,XY sex reversal and 46,XX lack of pubertal development as well as severe hypertension

Non-Virilizing Congenital Adrenal Hyperplasia in a Female Patient with a Novel HSD3B2 Mutation.

Classic 3β-hydroxysteroid dehydrogenase type 2 (3β-HSD II) deficiency causes congenital adrenal hyperplasia with glucocorticoid, mineralocorticoid, and sex steroid deficiency. We present a female patient with congenital adrenal hyperplasia detected in newborn screening due to elevated 17OH-progesterone. Female external genitalia and non-measurable androgen levels elicited the suspicion of a defect early in the steroid cascade. Two loss-of-function HSD3B2 mutations (1 novel) were detected and confirmed in silico. We argue that in a girl with glucocorticoid and mineralocorticoid deficiency without virilization, 3β-HSD II deficiency is an important differential diagnosis. 17OH-progesterone may initially be elevated due to placental and peripheral activity of 3β-HSD I, whereas dehydroepiandrosterone may not be increased

Broad phenotypes in heterozygous NR5A1 46,XY patients with a disorder of sex development: an oligogenic origin?

SF-1/NR5A1 is a transcriptional regulator of adrenal and gonadal development. NR5A1 disease-causing variants cause disorders of sex development (DSD) and adrenal failure, but most affected individuals show a broad DSD/reproductive phenotype only. Most NR5A1 variants show in vitro pathogenic effects, but not when tested in heterozygote state together with wild-type NR5A1 as usually seen in patients. Thus, the genotype-phenotype correlation for NR5A1 variants remains an unsolved question. We analyzed heterozygous 46,XY SF-1/NR5A1 patients by whole exome sequencing and used an algorithm for data analysis based on selected project-specific DSD- and SF-1-related genes. The variants detected were evaluated for their significance in literature, databases and checked in silico using webtools. We identified 19 potentially deleterious variants (one to seven per patient) in 18 genes in four 46,XY DSD subjects carrying heterozygous NR5A1 disease-causing variants. We constructed a scheme of all these hits within the landscape of currently known genes involved in male sex determination and differentiation. Our results suggest that the broad phenotype in these heterozygous NR5A1 46,XY DSD subjects may well be explained by an oligogenic mode of inheritance, in which multiple hits, individually non-deleterious, may contribute to a DSD phenotype unique to each heterozygous SF-1/NR5A1 individual

LRH-1 May Rescue SF-1 Deficiency for Steroidogenesis: An in vitro and in vivo Study

Steroidogenic factor 1 (NR5A1/SF-1) mutations usually manifest in 46,XY individuals with variable degrees of disordered sex development and in 46,XX women with ovarian insufficiency. So far, there is no genotype-phenotype correlation. The broad spectrum of phenotype with NR5A1 mutations may be due to a second hit in a gene with similar function to NR5A1/SF-1. Liver receptor homologue-1 (LRH-1/NR5A2) might be a good candidate. We performed in vitro studies for the interplay between SF-1, LRH-1 and DAX-1, expression profiles in human steroidogenic tissues, and NR5A2 genetic studies in a cohort (11 patients, 8 relatives, 11 families) harboring heterozygote NR5A1/SF-1 mutations. LRH-1 isoforms transactivate the CYP17A1 and HSD3B2 promoters similarly to SF-1 and compensate for SF-1 deficiency. DAX-1 inhibits SF-1- and LRH-1-mediated transactivation. LRH-1 is found expressed in human adult and fetal adrenals and testes. However, no NR5A2/LRH-1 mutations were detected in 14 individuals with heterozygote NR5A1/SF-1 mutations. These findings demonstrate that in vitro LRH-1 can act like SF-1 and compensate for its deficiency. Expression of LRH-1 in fetal testis suggests a role in male gonadal development. However, as we found no NR5A2/LRH-1 mutations, the 'second genetic hit' in SF-1 patients explaining the broad phenotypic variability remains elusive

Mechanism of the Dual Activities of Human CYP17A1 and Binding to Anti-Prostate Cancer Drug Abiraterone Revealed by a Novel V366M Mutation Causing 17,20 Lyase Deficiency

The CYP17A1 gene regulates sex steroid biosynthesis in humans through 17α-hydroxylase/17,20 lyase activities and is a target of anti-prostate cancer drug abiraterone. In a 46, XY patient with female external genitalia, together with a loss of function mutation S441P, we identified a novel missense mutation V366M at the catalytic center of CYP17A1 which preferentially impaired 17,20 lyase activity. Kinetic experiments with bacterially expressed proteins revealed that V366M mutant enzyme can bind and metabolize pregnenolone to 17OH-pregnenolone, but 17OH-pregnenolone binding and conversion to dehydroepiandrosterone (DHEA) was impaired, explaining the patient's steroid profile. Abiraterone could not bind and inhibit the 17α-hydroxylase activity of the CYP17A1-V366M mutant. Molecular dynamics (MD) simulations showed that V366M creates a "one-way valve" and suggests a mechanism for dual activities of human CYP17A1 where, after the conversion of pregnenolone to 17OH-pregnenolone, the product exits the active site and re-enters for conversion to dehydroepiandrosterone. The V366M mutant also explained the effectiveness of the anti-prostate cancer drug abiraterone as a potent inhibitor of CYP17A1 by binding tightly at the active site in the WT enzyme. The V366M is the first human mutation to be described at the active site of CYP17A1 that causes isolated 17,20 lyase deficiency. Knowledge about the specificity of CYP17A1 activities is of importance for the development of treatments for polycystic ovary syndrome and inhibitors for prostate cancer therap