Genetic disruption of 21-hydroxylase in zebrafish causes interrenal hyperplasia

Congenital adrenal hyperplasia is a group of common inherited disorders leading to glucocorticoid deficiency. Most cases are caused by 21-hydroxylase deficiency (21OHD). The systemic consequences of imbalanced steroid hormone biosynthesis due to severe 21OHD remains poorly understood. Therefore, we have developed a zebrafish model for 21OHD, which focuses on the impairment of glucocorticoid biosynthesis. A single 21-hydroxylase gene (cyp21a2) is annotated in the zebrafish genome based on sequence homology. Our in silico analysis of the Cyp21a2 protein sequence suggests a sufficient degree of similarity for the usage of zebrafish cyp21a2 to model aspects of human 21OHD in vivo. We determined the spatio-temporal expression patterns of cyp21a2 by whole mount in situ hybridisation and RT-PCR throughout early development. Early cyp21a2 expression is restricted to the interrenal gland (zebrafish adrenal counterpart) and the brain. To further explore the in vivo consequences of 21-hydroxylase deficiency we created several cyp21a2 null-allele zebrafish lines employing a transcription activator-like effector nuclease genomic engineering strategy. Homozygous mutant zebrafish larvae showed an upregulation of the hypothalamic-pituitary-interrrenal axis and interrenal hyperplasia. Furthermore, Cyp21A2-deficient larvae had a typical steroid profile with reduced concentrations of cortisol and increased concentrations of 17-hydroxyprogesterone and 21-deoxycortisol. Affected larvae showed an upregulation of the hypothalamic-pituitary-interrrenal axis and interrenal hyperplasia. Downregulation of the glucocorticoid-responsive genes pck1 and fkbp5 indicated systemic glucocorticoid deficiency. Our work demonstrates the crucial role of Cyp21a2 in glucocorticoid biosynthesis in zebrafish larvae and establishes a novel in vivo model allowing for studies of systemic consequences of altered steroid hormone synthesis

Biallelic and monoallelic ESR2 variants associated with 46,XY disorders of sex development

Purpose: Disorders or differences of sex development (DSDs) are rare congenital conditions characterized by atypical sex development. Despite advances in genomic technologies, the molecular cause remains unknown in 50% of cases. Methods: Homozygosity mapping and whole-exome sequencing revealed an ESR2 variant in an individual with syndromic 46, XY DSD. Additional cases with 46, XY DSD underwent whole-exome sequencing and targeted next-generation sequencing of ESR2. Functional characterization of the identified variants included luciferase assays and protein structure analysis. Gonadal ESR2 expression was assessed in human embryonic data sets and immunostaining of estrogen receptor-beta (ER-beta) was performed in an 8-week-old human male embryo. Results: We identified a homozygous ESR2 variant, c.541_543del p. (Asn181del), located in the highly conserved DNA-binding domain of ER-beta, in an individual with syndromic 46, XY DSD. Two additional heterozygous missense variants, c.251G>T p.(Gly84Val) and c.1277T>G p.(Leu426Arg), located in the N-terminus and the ligand-binding domain of ER-beta, were found in unrelated, nonsyndromic 46, XY DSD cases. Significantly increased transcriptional activation and an impact on protein conformation were shown for the p.(Asn181del) and p.(Leu426Arg) variants. Testicular ESR2 expression was previously documented and ER-beta immunostaining was positive in the developing intestine and eyes. Conclusion: Our study supports a role for ESR2 as a novel candidate gene for 46, XY DSD

A giant ovarian cyst in a neonate with classical 21-hydroxylase deficiency with very high testosterone levels demonstrating a high-dose hook effect

Congenital adrenal hyperplasia (CAH) is a group of disorders affecting the adrenal steroid synthesis. The most common form, 21-hydroxylase deficiency (21-OHD), leads to decreased production of cortisol and aldosterone with increased androgen secretion. In classic CAH, glucocorticoid treatment can be life-saving and serves to bring the symptoms under control. However, the treatment challenge is to effectively control the excess androgen effect by using the lowest possible glucocorticoid dose. Previous studies suggested a relationship between ovarian cyst formation and adrenal androgen excess, but neonatal large ovarian cysts have been very rarely reported in newborns with CAH. Here, we present the unique case of a neonate with classical 21-OHD who underwent surgery for a giant (10x8x7 cm) unilateral solitary ovarian follicular cyst on the 2nd postnatal day. Hormonal evaluation of the patient revealed high-dose hook effect for serum testosterone levels for the first time by a two-site immunoradiometric assay. Possible mechanisms by which androgen excess may cause ovarian cyst formation are discussed. Conflict of interest:None declared

Effect of maternal factors and fetomaternal glucose homeostasis on birth weight and postnatal growth

OBJECTIVE: It is important to identify the possible risk factors for the occurrence of large for gestational age (LGA) in newborns and to determine the effect of birth weight and metabolic parameters on subsequent growth. We aimed to determine the effects of maternal weight, weight gain during pregnancy, maternal hemoglobin A1c (HbA1c), C-peptide and insulin as well as cord C-peptide and insulin levels on birth weight and postnatal growth during the first two years of life. METHODS: Healthy, non-diabetic mothers and term singleton newborns were included in this prospective case-control cohort study. Fasting maternal glucose, HbA1c, C-peptide and insulin levels were studied. Cord blood was analyzed for C-peptide and insulin. At birth, newborns were divided into two groups according to birth size: LGA and appropriate for GA (AGA). Infants were followed at six-month intervals for two years and their length and weight were recorded. RESULTS: Forty LGA and 43 AGA infants were included in the study. Birth weight standard deviation score (SDS) was positively correlated with maternal body mass index (BMI) before delivery (r=0.2, p=0.04) and with weight gain during pregnancy (r=0.2, p=0.04). In multivariate analyses, the strongest association with macrosomia was a maternal C-peptide level >3.85 ng/mL (OR=20). Although the LGA group showed decreased growth by the 6-month of follow-up, the differences between the LGA and AGA groups in weight and length SDS persisted over the 2 years of follow-up. CONCLUSION: The control of maternal BMI and prevention of overt weight gain during pregnancy may prevent excessive birth weight. The effect of the in utero metabolic environment on the weight and length SDS of infants born LGA persists until at least two years of age

Insights From Long-term Follow-up of a Girl With Adrenal Insufficiency and Sphingosine-1-Phosphate Lyase Deficiency.

Introduction: Sphingosine-1-phosphate lyase (SGPL1) insufficiency syndrome (SPLIS) is a multisystemic disorder which, in the main, incorporates steroid-resistant nephrotic syndrome and primary adrenal insufficiency (PAI). Case Presentation: We present a young girl with a novel homozygous variant in SGPL1, p.D350G, with PAI in the absence of nephrotic syndrome. In the course of 15 years of follow-up she has further developed primary hypothyroidism and while she has progressed through puberty appropriately, ovarian calcifications were noted on imaging. The p.D350G variant results in reduced protein expression of SGPL1. We demonstrate that CRISPR engineered knockout of SGPL1 in human adrenocortical (H295R) cells abrogates cortisol production. Furthermore, while wild-type SGPL1 is able to rescue cortisol production in this in vitro model of adrenal disease, this is not observed with the p.D350G mutant. Conclusion: SGPL1 deficiency should be considered in the differential diagnosis of PAI with close attention paid to evolving disease on follow-up