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Introduction Patients with congenital hyperinsulinism due to ABCC8 variants generally present severe hypoglycaemia and those who do not respond to medical treatment typically undergo pancreatectomy. Few data exist on the natural history of non-pancreatectomised patients. This work aims to describe the genetic characteristics and natural history in a cohort of non-pancreatectomised patients with congenital hyperinsulinism due to variants in the ABCC8 gene. Subjects and Methods Ambispective study of patients with congenital hyperinsulinism with pathogenic or likely pathogenic variants in ABCC8 treated in the last 48 years and who were non-pancreatectomised. Continuous Glucose Monitoring (CGM) has been periodically performed in all patients since 2003. An oral glucose tolerance test (OGTT) was performed if hyperglycemia was detected in the CGM. Results Eighteen non-pancreatectomised patients with ABCC8 variants were included. Seven (38.9%) patients were heterozygous, eight (44.4%) compound heterozygous, two (11.1%) homozygous, and one patient carried two variants with incomplete familial segregation studies. Seventeen patients were followed-up and twelve (70.6%) of them evolved to spontaneous resolution (median age 6.0±4years; range:1-14). Five out of these twelve patients (41.7%) subsequently progressed to diabetes with insufficient insulin secretion. Evolution to diabetes was more frequent in patients with biallelic variants in the ABCC8 gene. Conclusion The high remission rate observed in our cohort makes conservative medical treatment a reliable strategy for the management of patients with congenital hyperinsulinism due to ABCC8 variants. In addition, a periodic follow-up of glucose metabolism after remission is recommended as a significant proportion of patients evolved to impaired glucose tolerance or diabetes (biphasic phenotype).

Steroidogenic factor 1 (SF-1,NR5A1) plays an important role in human sex development. Variants ofNR5A1/SF-1 may cause mild to severe differences of sex development (DSD) or may be found in healthy carriers. So far, the broad DSD phenotypic variability associatedNR5A1/SF-1 variants remains a conundrum. TheNR5A1/SF-1 variant c.437G>C/p.Gly146Ala is common in individuals with a DSD and has been suggested to act as a susceptibility factor for adrenal disease or cryptorchidism. However, as the allele frequency in the general population is high, and as functional testing of the p.Gly146Ala variantin vitrorevealed inconclusive results, the disease-causing effect of this variant has been questioned. However, a role as a disease modifier in concert with other gene variants is still possible given that oligogenic inheritance has been described in patients withNR5A1/SF-1 gene variants. Therefore, we performed next generation sequencing in DSD individuals harboring theNR5A1/SF-1 p.Gly146Ala variant to search for other DSD-causing variants. Aim was to clarify the function of this variant for the phenotype of the carriers. We studied 14 pediatric DSD individuals who carried the p.Gly146Ala variant. Panel and whole-exome sequencing was performed, and data were analyzed with a specific data filtering algorithm for detecting variants inNR5A1- and DSD-related genes. The phenotype of the studied individuals ranged from scrotal hypospadias and ambiguous genitalia in 46,XY DSD to typical male external genitalia and ovotestes in 46,XX DSD patients. Patients were of African, Spanish, and Asian origin. Of the 14 studied subjects, five were homozygous and nine heterozygous for theNR5A1/SF-1 p.Gly146Ala variant. In ten subjects we identified either a clearly pathogenic DSD gene variant (e.g. inAR, LHCGR) or one to four potentially deleterious variants that likely explain the observed phenotype alone (e.g. inFGFR3, CHD7, ADAMTS16). Our study shows that most individuals carrying theNR5A1/SF-1 p.Gly146Ala variant, harbor at least one other deleterious gene variant which can explain the DSD phenotype. This finding confirms that the p.Gly146Ala variant ofNR5A1/SF-1may not contribute to the pathogenesis of DSD and qualifies as a benign polymorphism. Thus, individuals, in whom theNR5A1/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 next-generation sequencing method to reveal the real genetic diagnosis.

Resumen Introduccion La discinesia ciliar primaria (DCP) es una enfermedad caracterizada por una alteracion en la estructura ciliar que impide el correcto aclaramiento de las secreciones respiratorias. Su diagnostico es complejo y se basa en una combinacion de tecnicas. El objetivo de este estudio fue disenar un panel de genes incluyendo todos los genes causantes conocidos y comprobar su utilidad diagnostica en una cohorte de pacientes espanoles. Metodos Estudio transversal multicentrico de pacientes con sospecha elevada de DCP, aplicando los criterios de la European Respiratory Society. Diseno de un panel de genes para secuenciacion masiva con la tecnologia de captura SeqCap EZ technology, incluyendo 44 genes relacionados con la DCP. Resultados Se incluyo a 79 pacientes de los que 53 presentaron un diagnostico de DCP confirmado o muy probable. La sensibilidad del panel de genes fue del 81,1% con una especificidad del 100%. Se encontraron variantes candidatas en alguno de los genes del panel en 43 de los pacientes con DCP, siendo 51,2% (22/43) homocigotos y 48,8% (21/43) heterocigotos compuestos. Los genes causales mas frecuentes fueron DNAH5 y CCDC39. Encontramos 52 variantes distintas, 36 no descritas previamente en la literatura. Conclusiones El diseno y la implementacion de un panel de genes a medida tiene un alto rendimiento diagnostico genetico de la DCP, lo que permite conocer mejor la afectacion causal de estos pacientes y sentar las bases para futuros abordajes terapeuticos.

Introduction Primary ciliary dyskinesia (PCD) is characterized by an alteration in the ciliary structure causing difficulty in the clearance of respiratory secretions. Diagnosis is complex and based on a combination of techniques. The objective of this study was to design a gene panel including all known causative genes, and to corroborate their diagnostic utility in a cohort of Spanish patients. Methods This was a multicenter cross-sectional study of patients with a high suspicion of PCD according to European Respiratory Society criteria. We designed a gene panel for massive sequencing using SeqCap EZ capture technology that included 44 genes associated with PCD. Results We included 79 patients, 53 of whom had a diagnosis of confirmed or highly probable PCD. The sensitivity of the gene panel was 81.1%, with a specificity of 100%. Candidate variants were found in some of the genes of the panel in 43 patients with PCD, 51.2% (22/43) of whom were homozygotes and 48.8% (21/43) compound heterozygotes. The most common causative genes were DNAH5 and CCDC39. We found 52 different variants, 36 of which were not previously described in the literature. Conclusions The design and implementation of a tailored gene panel produces a high yield in the genetic diagnosis of PCD. This panel provides a better understanding of the causative factors involved in these patients and lays down the groundwork for future therapeutic approaches.

Purpose Thyroid dyshormonogenesis is a heterogeneous group of hereditary diseases produced by a total/partial blockage of the biochemical processes of thyroid-hormone synthesis and secretion. Paired box 8 (PAX8) is essential for thyroid morphogenesis and thyroid hormone synthesis. We aimed to identify PAX8 variants in patients with thyroid dyshormonogenesis and to analyze them with in vitro functional studies. Patients and Methods Nine pediatric patients with a eutopic thyroid gland were analyzed by the Catalan screening program for congenital hypothyroidism. Scintigraphies showed absent, low, or normal uptake. Only one patient had a hypoplastic gland. On reevaluation, perchlorate discharge test was negative or compatible with partial iodine-organization deficit. After evaluation, 8 patients showed permanent mild or severe hypothyroidism. Massive-sequencing techniques were used to detect variants in congenital hypothyroidism-related genes. In vitro functional studies were based on transactivating activity of mutant PAX8 on a TG-gene promoter and analyzed by a dual-luciferase assays. Results We identified 7 heterozygous PAX8 exonic variants and 1 homozygous PAX8 splicing variant in 9 patients with variable phenotypes of thyroid dyshormonogenesis. Five were novel and 5 variants showed a statistically significant impaired transcriptional activity of TG promoter: 51% to 78% vs the wild type. Conclusions Nine patients presented with PAX8 candidate variants. All presented with a eutopic thyroid gland and 7 had deleterious variants. The phenotype of affected patients varies considerably, even within the same family; but, all except the homozygous patient presented with a normal eutopic thyroid gland and thyroid dyshormonogenesis. PAX8 functional studies have shown that 6 PAX8 variants are deleterious. Our studies have proven effective in evaluating these variants.

Context Mutations in cytochrome P450 oxidoreductase (POR) cause a form of congenital adrenal hyperplasia (CAH). We report a novel R550W mutation in POR identified in a 46,XX patient with signs of aromatase deficiency. Objective Analysis of aromatase deficiency from the R550W mutation in POR. Design, setting, and patient Both the child and the mother had signs of virilization. Ultrasound revealed the presence of uterus and ovaries. No defects in CYP19A1 were found, but further analysis with a targeted Disorders of Sexual Development NGS panel (DSDSeq.V1, 111 genes) on a NextSeq (Illumina) platform in Madrid and Barcelona, Spain, revealed compound heterozygous mutations c.73_74delCT/p.L25FfsTer93 and c.1648C > T/p.R550W in POR. Wild-type and R550W POR were produced as recombinant proteins and tested with multiple cytochrome P450 enzymes at University Children’s Hospital, Bern, Switzerland. Main outcome measure and Results POR-R550W showed 41% of the WT activity in cytochrome c and 7.7% activity for reduction of MTT. Assays of CYP19A1 showed a severe loss of activity, and CYP17A1 as well as CYP21A2 activities were also lost by more than 95%. Loss of CYP2C9, CYP2C19, and CYP3A4 activities was observed for the R550W-POR. Predicted adverse effect on aromatase activity as well as a reduction in binding of NADPH was confirmed. Conclusions Pathological effects due to POR-R550W were identified, expanding the knowledge of molecular pathways associated with aromatase deficiency. Screening of the POR gene may provide a diagnosis in CAH without defects in genes for steroid metabolizing enzymes.

[Background] Children's diffuse lung disease, also known as children's Interstitial Lung Diseases (chILD), are a heterogeneous group of rare diseases with relevant morbidity and mortality, which diagnosis and classification are very complex. Epidemiological data are scarce. The aim of this study was to analyse incidence and prevalence of chILD in Spain., [Methods] Multicentre observational prospective study in patients from 0 to 18 years of age with chILD to analyse its incidence and prevalence in Spain, based on data reported in 2018 and 2019., [Results] A total of 381 cases with chILD were notified from 51 paediatric pulmonology units all over Spain, covering the 91.7% of the paediatric population. The average incidence of chILD was 8.18 (CI 95% 6.28–10.48) new cases/million of children per year. The average prevalence of chILD was 46.53 (CI 95% 41.81–51.62) cases/million of children. The age group with the highest prevalence were children under 1 year of age. Different types of disorders were seen in children 2–18 years of age compared with children 0–2 years of age. Most frequent cases were: primary pulmonary interstitial glycogenosis in neonates (17/65), neuroendocrine cell hyperplasia of infancy in infants from 1 to 12 months (44/144), idiopathic pulmonary haemosiderosis in children from 1 to 5 years old (13/74), hypersensitivity pneumonitis in children from 5 to 10 years old (9/51), and scleroderma in older than 10 years old (8/47)., [Conclusions] We found a higher incidence and prevalence of chILD than previously described probably due to greater understanding and increased clinician awareness of these rare diseases., [Antecedentes] Las neumopatías intersticiales pediátricas, también conocidas con el acrónimo chILD (del inglés children's Interstitial Lung Diseases), es un grupo heterogéneo de enfermedades raras con morbimortalidad relevante, cuyo diagnóstico y clasificación son complejos. Los estudios epidemiológicos son escasos. El objetivo de este trabajo fue analizar la incidencia y la prevalencia de chILD en España., [Métodos] Estudio prospectivo observacional multicéntrico en pacientes de 0 a 18 años afectos de chILD para analizar la incidencia y la prevalencia en España, a partir de datos recogidos en 2018 y 2019., [Resultados] Se recogieron 381 casos de chILD entre 51 unidades de neumología pediátrica de toda España, que cubrían el 91,7% de la población pediátrica. La incidencia promedio fue 8,18 (IC 95%: 6,28-10,48) casos nuevos/millón de niños por año. La prevalencia promedio fue de 46,53 (IC 95%: 41,81-51,62) casos/millón de niños. El grupo de edad con mayor prevalencia fue el de niños menores de un año. Se observaron diferentes entidades en niños de 2 a 18 años en comparación con niños de 0 a 2 años. Los diagnósticos más frecuentes fueron: glucogenosis intersticial pulmonar primaria en neonatos (17/65), hiperplasia de células neuroendocrinas en lactantes de uno a 12 meses (44/144), hemosiderosis pulmonar idiopática en niños de uno a 5 años (13/74), neumonía por hipersensibilidad en niños de 5 a 10 años (9/51) y esclerodermia en mayores de 10 años (8/47)., [Conclusiones] Encontramos una mayor incidencia y prevalencia de chILD que las descritas previamente, probablemente debido a un mayor conocimiento y detección de estas enfermedades raras., ATV was supported by a grant from the Spanish Society of Paediatric Pulmonology and a Short Term Scientific Mission of the Cost CA 16125 ENTeR-chILD. This work was supported by a grant from the Spanish Society of Pneumology and Thoracic Surgery (SEPAR 2017/492). AMG was supported by a grant from the project HCQ4Surfdefect, in E-Rare-3, the ERA-Net for Research on Rare Diseases (Acciones complementarias en Salud, Instituto Carlos III, Madrid, Spain, AC16/00027) and Cost CA 16125 ENTeR-chILD.

Introduction Pulmonary interstitial glycogenosis (PIG) is a rare infant interstitial lung disease characterized by an increase in the number of interstitial mesenchymal cells, presenting as enhanced cytoplasmic glycogen, and is considered to represent the expression of an underlying lung development disorder. Methods This study describes the clinical, radiological, and functional characteristics and long-term outcomes (median 12 years) of nine infants diagnosed with isolated PIG associated with alveolar simplification in the absence of other diseases. Results All patients presented with tachypnea. Additionally, seven patients had breathing difficulties and hypoxemia. Abnormalities in chest-computerized tomography (CT) with a pattern of ground-glass opacity, septal thickening, and air trapping were observed in all individuals, with images suggesting abnormal alveolar growth (parenchymal bands and architectural distortion). All lung biopsies showed alveolar simplification associated with an increased number of interstitial cells, which appeared as accumulated cytoplasmic glycogen. In the follow-up, all patients were asymptomatic. The respiratory function test was normal in only two patients. Five children showed an obstructive pattern, and two children showed a restrictive pattern. Chest-CT, performed after an average of 6.5 years since the initial investigation, revealed a partial improvement of the ground-glass opacity pattern; however, relevant alterations persisted. Conclusion Although the patients with PIG in the absence of other associated pathologies had a good clinical outcome, significant radiographic alterations and sequelae in lung function were still observed after a median follow-up of 12 years, suggesting that PIG is a marker of some other persistent abnormalities in lung growth, which have effects beyond the symptomatic period.

Persistent müllerian duct syndrome (PMDS) is characterized by the presence of müllerian duct derivatives in otherwise phenotypically normal males. Homozygous or compound heterozygous alterations in AMH or AMHR2 have been identified in approximately 88% of PMDS cases. We report on a male patient with bilateral undescended gonads, müllerian derivatives, and normal serum AMH levels. A novel homozygous missense mutation, c.119G>C;p.Gly40Ala, in exon 2 of AMHR2 was detected that supported the clinical diagnosis of PMDS.

INTRODUCTION: Primary ciliary dyskinesia (PCD) is characterized by an alteration in the ciliary structure causing difficulty in the clearance of respiratory secretions. Diagnosis is complex and based on a combination of techniques. The objective of this study was to design a gene panel including all known causative genes, and to corroborate their diagnostic utility in a cohort of Spanish patients.; METHODS: This was a multicenter cross-sectional study of patients with a high suspicion of PCD, according to European Respiratory Society criteria, designed around a gene panel for massive sequencing using SeqCap EZ capture technology that included 44 genes associated with PCD.; RESULTS: We included 79 patients, 53 of whom had a diagnosis of confirmed or highly probable PCD. The sensitivity of the gene panel was 81.1%, with a specificity of 100%. Candidate variants were found in some of the genes of the panel in 43 patients with PCD, 51.2% (22/43) of whom were homozygotes and 48.8% (21/43) compound heterozygotes. The most common causative genes were DNAH5 and CCDC39. We found 52 different variants, 36 of which were not previously described in the literature.; CONCLUSIONS: The design and implementation of a tailored gene panel produces a high yield in the genetic diagnosis of PCD. This panel provides a better understanding of the causative factors involved in these patients and lays down the groundwork for future therapeutic approaches. Copyright © 2020 SEPAR. Publicado por Elsevier Espana, S.L.U. All rights reserved.

Background: Primary ciliary dyskinesia (PCD) is a disease characterized by an alteration in the ciliary structure that causes an abnormal clearance of respiratory secretions. Its diagnosis is complex and is based on a combination of different techniques. The objective of this study was to design a gene panel including all known caustive genes, and to verify the utility for diagnostic in a cohort of Spanish patients. Methods: Multicenter cross-sectional study of patients with high suspicion of PCD, applying the criteria of the European Respiratory Society. Design a gene panel for mass sequencing with SeqCap EZ technology, including 44 PCD-related genes. Results: 79 patients were included, 53 of those diagnosed with confirmed or highly likely PCD. The sensitivity of the gene panel was 81.1% with a specificity of 100%. Candidate variants were found in any of the panel genes 43 patients with PCD, with 51.2% (22/43) homozygous and 48.8% (21/43) compound heterozygous. The most common causal genes were DNAH5 and CCDC39, in patients of Caucasian origin, while in non-Caucasian ones the most common causal genes were CCDC40, DNAI2 and RSPH4A. We found 52 different variants, 36 not previously described in the literature. Conclusions: The results of this study show that the design and implementation of an specific gene panel has a high efficiency forgenetic diagnostic of PCD, allowing for a better understanding of the causal of this disease and laying the basis for future therapeutic approaches

Congenital adrenal hyperplasia (CAH) consists of several autosomal recessive disorders that inhibit steroid biosynthesis. We describe a case report diagnosed with adrenal insufficiency due to low adrenal steroids and adrenocorticotropic hormone excess due to lack of cortisol negative feedback signaling to the pituary gland. Genetic work up revealed two missense variants, p.Thr204Arg and p.Leu260Arg in the STAR gene, inherited by both parents (non-consanguineous). The StAR protein supports CYP11A1 enzyme to cleave the side chain of cholesterol and synthesize pregnenolone which is metabolized to all steroid hormones. We used bioinformatics to predict the impact of the variants on StAR activity and then we performed functional tests to characterize the two novel variants. In a cell system we tested the ability of variants to support cholesterol conversion to pregnenolone and measured their mRNA and protein expression. For both variants, we observed loss of StAR function, reduced protein expression and categorized them as pathogenic variants according to guidelines of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. These results fit the phenotype of the girl during diagnosis. This study characterizes two novel variants and expands the list of missense variants that cause CAH.

Context: Mutations in Cytochrome P450 oxidoreductase (POR) cause a form of congenital adrenal hyperplasia (CAH). We are reporting a novel R550W mutation in POR identified in a 46, XX patient with signs of aromatase deficiency. Objective: Analysis of aromatase deficiency from R550W mutation in POR. Design, Setting, and Patient: Both the child and the mother had signs of virilization. Ultrasound revealed the presence of uterus and ovaries. No defects in CYP19A1 were found, but further analysis with a targeted Disorders of Sexual Development NGS panel (DSDSeq.V1, 111 genes) on a NextSeq (Illumina) platform in Madrid and Barcelona, Spain, revealed compound heterozygous mutations c.73_74delCT/p.L25FfsTer93 and c.1648C>T/p.R550W in POR. WT and R550W POR were produced as recombinant proteins and tested with multiple cytochrome P450 enzymes at University Children’s Hospital, Bern, Switzerland. Main Outcome Measure and Results: R550W POR showed 41% of the WT activity in cytochrome c and 7.7% activity for reduction of MTT. Assays of CYP19A1 showed a severe loss of activity and CYP17A1, as well as CYP21A2 activities, were also lost by more than 95%. Loss of CYP2C9, CYP2C19, and CYP3A4 activities was observed for the R550W-POR. Predicted adverse effect on aromatase activity as well as a reduction in binding of NADPH was confirmed. Conclusions: Pathological effects due to POR R550W were identified, expanding the knowledge of molecular pathways associated with aromatase deficiency. Screening of the POR gene may provide a diagnosis in CAH without defects in genes for steroid metabolizing enzymes.

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.

Scientific knowledge to understand the biological basis of sex development was prompted by the observation of variants different from the 2 most frequent body types, and this became one of the fields first studied by modern pediatric endocrinology. The clinical observation was supported by professionals working in different areas of laboratory sciences which led to the description of adrenal and gonadal steroidogenesis, the enzymes involved, and the different deficiencies. Steroid hormone measurements evolved from colorimetry to radioimmunoassay (RIA) and automated immunoassays, although gas and liquid chromatography coupled to mass spectrometry are now the gold standard techniques for steroid measurements. Peptide hormones and growth factors were purified, and their measurement evolved from RIA to automated immunoassays. Hormone action mechanisms were described, and their specific receptors were characterized and assayed in experimental materials and in patient tissues and cell cultures. The discovery of the genetic basis for variant sex developments began with the description of the sex chromosomes. Molecular technology allowed cloning of genes coding for the different proteins involved in sex determination and development. Experimental animal models aided in verifying the roles of proteins and also suggested new genes to be investigated. New candidate genes continue to be described based on experimental models and on next-generation sequencing of patient DNAs.

Background: Primary ciliary dyskinesia (PCD) diagnosis is based on studying ciliary function by high-speed video-microscopy (HSVM) and ciliary ultrastructure by transmission electron microscopy (TEM). Immunofluorescence (IF) analysis has been proposed as a routine test to improve PCD diagnosis rate. We show a preliminary HSVM and IF results of PCD candidate patients Methods: A cohort of patients was studied because of high clinical suspicion of PCD. Samples of ciliated nasal epithelium were obtained by brushing. HSVM for analysing ciliary beat frequency (CBF, normal ≥9Hz) and pattern (CBP, normal ≤20% dyskinetic ciliated cells) was performed using a high-speed camera and optical microscope. Samples from 30 patients were prepared for IF analysis and labelled with a panel of 4 ciliary-ultrastructure antibodies: DNAH5, DNALI1, GAS8, RSPH4A Results: We present HSVM and IF results of 30 patients. HSVM analyses showed 22 (73%) patients presented a reduced CBF and 28 (93%) an altered CBP (12 had disorganised ciliary beat, 10 immotile cilia and 6 stiff cilia). Regarding IF analysis, 23 (77%) were evaluable for all antibodies: 14 were normal and 9 had one or moreabsent proteins (6 DNAH5, 1 DNALI1, 1 DNAH5 and DNALI1 and 1 DNAH5, DNALI1 and GAS8). All cases with a defect in IF presented a concordant CBP. Only one case with a normal IF had also a normal HSVM. The other 13 cases had a normal IF but an altered CBP, for which further studies are being performed Conclusion: The combination of HSVM and IF could explain an important number of cases, although other analyses will be necessary to increase the accuracy and diagnosis rate of PCD

Context: Mutations in Cytochrome P450 oxidoreductase (POR) cause a form of congenital adrenal hyperplasia (CAH). We are reporting a novel R550W mutation in POR identified in a 46, XX patient with signs of aromatase deficiency. Objective: Analysis of aromatase deficiency from R550W mutation in POR. Design, Setting, and Patient: Both the child and the mother had signs of virilization. Ultrasound revealed the presence of uterus and ovaries. No defects in CYP19A1 were found, but further analysis with a targeted Disorders of Sexual Development NGS panel (DSDSeq.V1, 111 genes) on a NextSeq (Illumina) platform in Madrid and Barcelona, Spain, revealed compound heterozygous mutations c.73_74delCT/p.L25FfsTer93 and c.1648C>T/p.R550W in POR. WT and R550W POR were produced as recombinant proteins and tested with multiple cytochrome P450 enzymes at University Children’s Hospital, Bern, Switzerland. Main Outcome Measure and Results: R550W POR showed 41% of the WT activity in cytochrome c and 7.7% activity for reduction of MTT. Assays of CYP19A1 showed a severe loss of activity and CYP17A1, as well as CYP21A2 activities, were also lost by more than 95%. Loss of CYP2C9, CYP2C19, and CYP3A4 activities was observed for the R550W-POR. Predicted adverse effect on aromatase activity as well as a reduction in binding of NADPH was confirmed. Conclusions: Pathological effects due to POR R550W were identified, expanding the knowledge of molecular pathways associated with aromatase deficiency. Screening of the POR gene may provide a diagnosis in CAH without defects in genes for steroid metabolizing enzymes.

Persistent müllerian duct syndrome (PMDS) is characterized by the presence of müllerian duct derivatives in otherwise phenotypically normal males. Homozygous or compound heterozygous alterations in AMH or AMHR2 have been identified in approximately 88% of PMDS cases. We report on a male patient with bilateral undescended gonads, müllerian derivatives, and normal serum AMH levels. A novel homozygous missense mutation, c.119GC;p.Gly40Ala, in exon 2 of AMHR2 was detected that supported the clinical diagnosis of PMDS.

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.

Key Clinical Message 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.

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.

Steroidogenic factor 1 (NR5A1/SF 1) mutations usually manifest in 46XY individuals with variable degrees of disordered sex development and in 46XX 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. © 2015 S. Karger AG Basel

Primary adrenal insufficiency is life threatening and can present alone or in combination with other comorbidities. Here, we have described a primary adrenal insufficiency syndrome and steroid-resistant nephrotic syndrome caused by loss-of-function mutations in sphingosine-1-phosphate lyase (SGPL1). SGPL1 executes the final decisive step of the sphingolipid breakdown pathway, mediating the irreversible cleavage of the lipid-signaling molecule sphingosine-1-phosphate (S1P). Mutations in other upstream components of the pathway lead to harmful accumulation of lysosomal sphingolipid species, which are associated with a series of conditions known as the sphingolipidoses. In this work, we have identified 4 different homozygous mutations, c.665G>A (p.R222Q), c.1633_1635delTTC (p.F545del), c.261+1G>A (p.S65Rfs*6), and c.7dupA (p.S3Kfs*11), in 5 families with the condition. In total, 8 patients were investigated, some of whom also manifested other features, including ichthyosis, primary hypothyroidism, neurological symptoms, and cryptorchidism. Sgpl1-/- mice recapitulated the main characteristics of the human disease with abnormal adrenal and renal morphology. Sgpl1-/- mice displayed disrupted adrenocortical zonation and defective expression of steroidogenic enzymes as well as renal histology in keeping with a glomerular phenotype. In summary, we have identified SGPL1 mutations in humans that perhaps represent a distinct multisystemic disorder of sphingolipid metabolism. ispartof: Journal of Clinical Investigation vol:127 issue:3 pages:942-953 ispartof: location:United States status: published

OBJECTIVE The steroidogenic acute regulatory protein (StAR) transports cholesterol to the mitochondria for steroidogenesis. Loss of StAR function causes lipoid congenital adrenal hyperplasia (LCAH) which is characterized by impaired synthesis of adrenal and gonadal steroids causing adrenal insufficiency, 46,XY disorder of sex development (DSD) and failure of pubertal development. Partial loss of StAR activity may cause adrenal insufficiency only. PATIENT A newborn girl was admitted for mild dehydration, hyponatremia, hyperkalemia and hypoglycaemia and had normal external female genitalia without hyperpigmentation. Plasma cortisol, 17OH-progesterone, DHEA-S, androstendione and aldosterone were low, while ACTH and plasma renin activity were elevated, consistent with the diagnosis of primary adrenal insufficiency. Imaging showed normal adrenals, and cytogenetics revealed a 46,XX karyotype. She was treated with fluids, hydrocortisone and fludrocortisone. DESIGN, METHODS AND RESULTS Genetic studies revealed a novel homozygous STAR mutation in the 3' acceptor splice site of intron 4, c.466-1G>A (IVS4-1G>A). To test whether this mutation would affect splicing, we performed a minigene experiment with a plasmid construct containing wild-type or mutant StAR gDNA of exons-introns 4-6 in COS-1 cells. The splicing was assessed on total RNA using RT-PCR for STAR cDNAs. The mutant STAR minigene skipped exon 5 completely and changed the reading frame. Thus, it is predicted to produce an aberrant and shorter protein (p.V156GfsX19). Computational analysis revealed that this mutant protein lacks wild-type exons 5-7 which are essential for StAR-cholesterol interaction. CONCLUSIONS STAR c.466-1A skips exon 5 and causes a dramatic change in the C-terminal sequence of the protein, which is essential for StAR-cholesterol interaction. This splicing mutation is a loss-of-function mutation explaining the severe phenotype of our patient. Thus far, all reported splicing mutations of STAR cause a severe impairment of protein function and phenotype.

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.

There is a controversy regarding the effects of the analogues of the gonadotrophin-releasing hormone (GnRH) in radiotherapy. This has led us to study the possible radio-protection of the ovarian function of a GnRH agonist analogue (GnRHa), triptorelin. in adult, female rats (Rattus norvegicus sp) The effects of the X-irradiation on the oocytes of ovarian primordial follicles, with and without GnRHa treatment, were compared, directly in the female rats (F-0) with reproductive parameters, and in the somatic cells of the resulting foetuses (F-1) with cytogenetical parameters. In order to do this. the ovaries and uteri from 82 females were extracted for the reproductive analysis and 236 foetuses were obtained for cytogenetical analysis The cytogenetical study was based on the data from 22,151 metaphases analysed The cytogenetical parameters analysed to assess the existence of chromosomal instability were the number of aberrant metaphases (2234) and the number (2854) and type of structural chromosomal aberrations, including gaps and breaks. Concerning the reproductive analysis of the ovaries and the uteri, the parameters analysed were the number of corpora lutea, implantations, implantation losses and foetuses Triptorelin confers radio-protection of the ovaries in front of chromosomal instability. which is different, with respect to the single and fractioned dose The cytogenetical analysis shows a general decrease in most of the parameters of the triptorelin-treated groups. with respect to their controls, and some of these differences were considered to be statistically significant The reproductive analysis indicates that there is also radio-protection by the agonist, although minor to the cytogenetical one. Only some of the analysed parameters show a statistically significant decrease in the triptorelin-treated groups (C) 2009 Elsevier B V All rights reserved

Background: Congenital malformations are present in approximately 2–3% of liveborn babies and 20% of stillborn fetuses. The mechanisms underlying the majority of sporadic and isolated congenital malformations are poorly understood, although it is hypothesized that the accumulation of rare genetic, genomic and epigenetic variants converge to deregulate developmental networks. Methodology/Principal Findings: We selected samples from 95 fetuses with congenital malformations not ascribed to a specific syndrome (68 with isolated malformations, 27 with multiple malformations). Karyotyping and Multiplex Ligation-dependent Probe Amplification (MLPA) discarded recurrent genomic and cytogenetic rearrangements. DNA extracted from the affected tissue (46%) or from lung or liver (54%) was analyzed by molecular karyotyping. Validations and inheritance were obtained by MLPA. We identified 22 rare copy number variants (CNV) [>100 kb, either absent (n = 7) or very uncommon (n = 15

SummaryObjective Molecular causes of isolated severe growth hormone deficiency (ISGHD) in several genes have been established. The aim of this study was to analyse the contribution of growth hormone-releasing hormone receptor (GHRHR) gene sequence variation to GH deficiency in a series of prepubertal ISGHD patients and to normal adult height. Design, subjects and measurements A systematic GHRHR gene sequence analysis was performed in 69 ISGHD patients and 60 normal adult height controls (NAHC). Four GHRHR single-nucleotide polymorphisms (SNPs) were genotyped in 248 additional NAHC. An analysis was performed on individual SNPs and combined genotype associations with diagnosis in ISGHD patients and with height-SDS in NAHC. Results Twenty-one SNPs were found. P3, P13, P15 and P20 had not been previously described. Patients and controls shared 12 SNPs (P1, P2, P4–P11, P16 and P21). Significantly different frequencies of the heterozygous genotype and alternate allele were detected in P9 (exon 4, rs4988498) and P12 (intron 6, rs35609199); P9 heterozygous genotype frequencies were similar in patients and the shortest control group (heights between −2 and −1 SDS) and significantly different in controls (heights between −1 and +2 SDS). GHRHR P9 together with 4 GH1 SNP genotypes contributed to 6·2% of height-SDS variation in the entire 308 NAHC. Conclusions This study established the GHRHR gene sequence variation map in ISGHD patients and NAHC. No evidence of GHRHR mutation contribution to ISGHD was found in this population, although P9 and P12 SNP frequencies were significantly different between ISGHD and NAHC. Thus, the gene sequence may contribute to normal adult height, as demonstrated in NAHC.

Background Among neotropical Primates, the Cai monkey Cebus paraguayanus (CPA) presents long, conserved chromosome syntenies with the human karyotype (HSA) as well as numerous C+ blocks in different chromosome pairs. In this study, immunofluorescence (IF) against two proteins of the Synaptonemal Complex (SC), namely REC8 and SYCP1, two recombination protein markers (RPA and MLH1), and one protein involved in the pachytene checkpoint machinery (BRCA1) was performed in CPA spermatocytes in order to analyze chromosome meiotic behavior in detail. Results Although in the vast majority of pachytene cells all autosomes were paired and synapsed, in a small number of nuclei the heterochromatic C-positive terminal region of bivalent 11 remained unpaired. The analysis of 75 CPA cells at pachytene revealed a mean of 43.22 MLH1 foci per nucleus and 1.07 MLH1 foci in each CPA bivalent 11, always positioned in the region homologous to HSA chromosome 21. Conclusion Our results suggest that C blocks undergo delayed pairing and synapsis, although they do not interfere with the general progress of pairing and synapsis.

Genomic instability can be produced by ionising radiation, so-called radiation-induced genomic instability, and chemical mutagens. Radiation-induced genomic instability occurs in both germinal and somatic cells and also in the offspring of irradiated individuals, and it is characterised by genetic changes including chromosomal rearrangements. The majority of studies of trans-generational, radiation-induced genomic instability have been described in the male germ line, whereas the authors who have chosen the female as a model are scarce. The aim of this work is to find out the radiation-induced effects in the foetal offspring of X-ray-treated female rats and, at the same time, the possible impact of this radiation-induced genomic instability on the action of a chemical mutagen. In order to achieve both goals, the quantity and quality of chromosomal damage were analysed. In order to detect trans-generational genomic instability, a total of 4806 metaphases from foetal tissues from the foetal offspring of X-irradiated female rats (5 Gy, acute dose) were analysed. The study's results showed that there is radiation-induced genomic instability: the number of aberrant metaphases and the breaks per total metaphases studied increased and were found to be statistically significant ( p ≤ 0.05), with regard to the control group. In order to identify how this trans-generational, radiation-induced chromosomal instability could influence the chromosomal behaviour of the offspring of irradiated rat females in front of a chemical agent (aphidicolin), a total of 2481 metaphases were studied. The observed results showed that there is an enhancement of the action of the chemical agent: chromosomal breaks per aberrant metaphases show significant differences ( p ≤ 0.05) in the X-ray- and aphidicolin-treated group as regards the aphidicolin-treated group. In conclusion, our findings indicate that there is trans-generational, radiation-induced chromosomal instability in the foetal cells from X-ray-treated female rats and that this RIGI enhances the chromosomal damage caused by the chemical agent aphidicolin.