Novel Genomic Variants, Atypical Phenotypes and Evidence of a Digenic/Oligogenic Contribution to Disorders/Differences of Sex Development in a Large North African Cohort.

In a majority of individuals with disorders/differences of sex development (DSD) a genetic etiology is often elusive. However, new genes causing DSD are routinely reported and using the unbiased genomic approaches, such as whole exome sequencing (WES) should result in an increased diagnostic yield. Here, we performed WES on a large cohort of 125 individuals all of Algerian origin, who presented with a wide range of DSD phenotypes. The study excluded individuals with congenital adrenal hypoplasia (CAH) or chromosomal DSD. Parental consanguinity was reported in 36% of individuals. The genetic etiology was established in 49.6% (62/125) individuals of the total cohort, which includes 42.2% (35/83) of 46, XY non-syndromic DSD and 69.2% (27/39) of 46, XY syndromic DSD. No pathogenic variants were identified in the 46, XX DSD cases (0/3). Variants in the AR, HSD17B3, NR5A1 and SRD5A2 genes were the most common causes of DSD. Other variants were identified in genes associated with congenital hypogonadotropic hypogonadism (CHH), including the CHD7 and PROKR2. Previously unreported pathogenic/likely pathogenic variants (n = 30) involving 25 different genes were identified in 22.4% of the cohort. Remarkably 11.5% of the 46, XY DSD group carried variants classified as pathogenic/likely pathogenic variant in more than one gene known to cause DSD. The data indicates that variants in PLXNA3, a candidate CHH gene, is unlikely to be involved in CHH. The data also suggest that NR2F2 variants may cause 46, XY DSD.

Genotype, Mortality, Morbidity, and Outcomes of 3ß-Hydroxysteroid Dehydrogenase Deficiency in Algeria.

Background: 3ß-hydroxysteroid dehydrogenase 2 (3ßHSD2) deficiency is a rare form of congenital adrenal hyperplasia (CAH), with fewer than 200 cases reported in the world literature and few data on outcomes. Patients and Methods: We report a mixed longitudinal and cross-sectional study from a single Algerian center between 2007 and 2021. Virilization and under-masculinization were assessed using Prader staging and the external masculinization score (EMS), pubertal development staged according to the system of Tanner. Adrenal steroids were measured using mass spectrophotometry (LC-MS/MS). A genetic analysis of HSD3B2 was performed using Sanger sequencing. Results: A 3ßHSD2 defect was confirmed in 6 males and 8 females from 10 families (8 consanguineous), with p.Pro222Gln mutation in all but two siblings with a novel deletion: c.453_464del or p.(Thr152_Pro155del). Probable 3ßHSD2 deficiency was diagnosed retrospectively in a further 6 siblings who died, and in two patients from two other centers. In the genetically confirmed patients, the median (range) age at presentation was 20 (0-390) days, with salt-wasting (n = 14) and genital anomaly (n = 10). The Prader stage for female patients was 2 (1-2) with no posterior fusion of the labia. The EMS for males was 6 (3-9). Median (range) values at diagnosis for 17-hydroxyprogesterone (17-OHP), dehydroepiandrosterone sulfate (DHEA-S), and 17-hydroxypregnenolone (17OHPreg) were elevated: 73.7 (0.37-164.3) nmol/L; 501.2(9.4-5441.3) nmol/L, and 139.7 (10.9-1500) nmol/l (NB >90 nmol/L diagnostic of 3ßHSD2 defect). Premature pubarche was observed in four patients (3F:1M). Six patients (5F:1M) entered puberty spontaneously, aged 11 (5-13) years in 5 girls and 11.5 years in one boy. Testicular adrenal rest tumors were found in three boys. Four girls reached menarche at 14.3 (11-14.5) years, with three developing adrenal masses (surgically excised in two) and polycystic ovary syndrome (PCOS), with radiological evidence of ovarian adrenal rest tumor in one. The median IQ was 90 (43-105), >100 in only two patients and <70 in three. Conclusions: The prevalence of 3ßHSD2 deficiency in Algeria appears high, with p.Pro222Gln being the most frequent mutation. Mortality is also high, with significant morbidity from adrenal tumors and PCOS in adolescence and an increased risk of learning disability. The finding of adrenal tumors in older patients with 3ßHSD2 indicates under-replacement, requiring effective hydrocortisone and fludrocortisone treatment rather than surgical removal.

Access to fludrocortisone and to hydrocortisone in children with congenital adrenal hyperplasia in the WHO Eastern Mediterranean Region: it takes a village .

Congenital adrenal hyperplasia (CAH), if untreated, carries high morbidity and mortality. A higher incidence of CAH is expected in countries where consanguinity is common, such as in the countries of the WHO Eastern Mediterranean Region (EMRO). CAH is managed through lifelong treatment with fludrocortisone and hydrocortisone. In this analysis, performed in the 22 EMRO countries and territories plus Algeria, we review which countries offer a neonatal screening programme for CAH and describe the barriers and opportunities to access oral fludrocortisone and oral and injectable hydrocortisone. Neonatal CAH screening was only available nationally in Qatar, Kuwait and partially in Lebanon and Saudi Arabia. We reviewed the national lists of essential medicines (NEMLs) and found that 13/23 (57%) countries included fludrocortisone and 18/23 (78%) included oral hydrocortisone. Fludrocortisone was not included by any of the low-income countries and oral hydrocortisone was only included by one low-income country. We then contacted paediatric endocrinologists in each country to assess perceived availability of these medicines. Overall, there was a relatively good consistency between inclusion of fludrocortisone and hydrocortisone in the NEML and their actual availability in a country. We propose several mechanisms to improve access, including prequalification by the WHO, a common registration process for groups of countries, pooled procurement, working with local pharmaceutical companies, special access status for medicines not yet registered in a country and compounding. We suggest that access to medicines requires a collaboration between health professionals, families of patients, health authorities, pharmaceutical companies and the WHO.

Expanding DSD Phenotypes Associated with Variants in the DEAH-Box RNA Helicase DHX37.

Missense variants in the RNA-helicase DHX37 are associated with either 46,XY gonadal dysgenesis or 46,XY testicular regression syndrome (TRS). DHX37 is required for ribosome biogenesis, and this subgroup of XY DSD is a new human ribosomopathy. In a cohort of 140 individuals with 46,XY DSD, we identified 7 children with either 46,XY complete gonadal dysgenesis or 46,XY TRS carrying rare or novel DHX37 variants. A novel p.R390H variant within the RecA1 domain was identified in a girl with complete gonadal dysgenesis. A paternally inherited p.R487H variant, previously associated with a recessive congenital developmental syndrome, was carried by a boy with a syndromic form of 46,XY DSD. His phenotype may be explained in part by a novel homozygous loss-of-function variant in the NGLY1 gene, which causes a congenital disorder of deglycosylation. Remarkably, a homozygous p.T477H variant was identified in a boy with TRS. His fertile father had unilateral testicular regression with typical male genital development. This expands the DSD phenotypes associated with DHX37. Structural analysis of all variants predicted deleterious effects on helicase function. Similar to all other known ribosomopathies, the mechanism of pathogenesis is unknown.

Novel CYP19A1 Mutations Extend the Genotype-Phenotype Correlation and Reveal the Impact on Ovarian Function.

CONTEXT: The steroidogenic enzyme aromatase (CYP19A1) is required for estrogen biosynthesis from androgen precursors in the ovary and extragonadal tissues. The role of aromatase, and thus estrogens, is best illustrated by genetic variations of the CYP19A1 gene leading to aromatase deficiency or excess. OBJECTIVE: The objective of this work is to characterize novel CYP19A1 variants. DESIGN SETTING AND PATIENTS: Variants causing aromatase deficiency were suspected in four 46,XX children of African and Indian origin by careful clinical phenotyping. Sequencing of the CYP19A1 gene identified novel variants. Minigene experiments, aromatase activity assay, and computational, and histological analysis were used to characterize the variants. MAIN OUTCOME MEASURE AND RESULTS: CYP19A1 variants were found in all patients: a deletion in intron 9 leading to p.P423_H503del, a delins variant at p.P154, and point variants p.V161D, p.R264C, p.R375C. Except for R264C, all variants showed a loss of function. Protein structure and dynamics studies were in line with functional assays. The 2 female patients with delins variants manifested with ambiguous genitalia at birth. Histologic investigation revealed normal ovarian tissue on one side and a streak gonad on the other. Two female patients presented with abnormal pubertal development and polycystic ovaries. CONCLUSION: In girls, aromatase deficiency usually manifests at birth, but diagnosis may also be made because of abnormal pubertal development or ovarian torsion due to (poly)cystic ovaries. The ovary harboring CYP19A1 variants may present as streak gonad or appears normal at birth, but is then at very high risk to produce cysts with aging and is therefore prone to ovarian torsion.

Primary gonadal failure.

The term primary gonadal failure encompasses not only testicular insufficiency in 46,XY males and ovarian insufficiency in 46,XX females, but also those disorders of sex development (DSD) which result in gender assignment that is at variance with the genotype and gonadal type. In boys, causes of gonadal failure include Klinefelter and other aneuploidy syndromes, bilateral cryptorchidism, testicular torsion, and forms of 46,XY DSD such as partial androgen insensitivity. Causes in girls include Turner syndrome and other aneuploidies, galactosemia, and autoimmune ovarian failure. Iatrogenic causes in both boys and girls include the late effects of childhood cancer treatment, total body irradiation prior to bone marrow transplantation, and iron overload in transfusion-dependent thalassaemia. In this paper, a brief description of the physiology of testicular and ovarian development is followed by a section on the causes and practical management of gonadal impairment in boys and girls. Protocols for pubertal induction and post-pubertal hormone replacement - intramuscular, oral and transdermal testosterone in boys; oral and transdermal oestrogen in girls - are then given. Finally, current and future strategies for assisted conception and fertility preservation are discussed.

Sex assignment practice in disorders of sexual differentiation: survey results from paediatric endocrinologists in the Arab region.

Background Sex assignment is a major issue in disorders of sexual differentiation (DSD). Not all conditions of DSD have clear recommendations on assignment and timing of surgery. Reports about sex assignment practice and the influence of culture and religion in the Arab region are scarce. Methods A survey questionnaire was distributed to participants in a paediatric endocrinology conference. Four DSD cases were presented. Participants were asked to fill in their answers on sex assignment choice, reasons for the particular assignment, strength of own recommendation and timing of surgery based on their practice. The cases presented were severely virilised XX congenital adrenal hyperplasia (CAH), complete androgen insensitivity syndrome (CAIS), severely undervirilised 5α reductase deficiency (5α RD) and XX ovotesticular case. Results Eighty-five endocrinologists participated in the study. Eighty (97.5%) chose a female sex to assign for the XX CAH. For the CAIS, 64 (78%) chose a female sex. Seventy-one (86.5%) voted for a male sex for the XY case of 5α RD. Forty-seven (57%) and 35 (43%) chose a female and a male sex for the ovotesticular case, respectively. The majority indicated that their advice for sex assignment is based on strong recommendations for the CAH, CAIS and 5α RD patients but they were open to the parents' cultural and religious beliefs in their decision of the assignment for the ovotesticular case. Conclusions Practice in the Arab region appears to be in line with the international guidelines in the majority of DSD sex assignment and timing of surgery issues. However, culture and religious beliefs influence the practice in certain circumstances.

Familial Multiplicity of Estrogen Insensitivity Associated With a Loss-of-Function ESR1 Mutation.

Context: Estrogens influence many physiological processes in mammals, including reproduction. Estrogen peripheral actions are mainly mediated through estrogen receptors (ERs) α and ß, encoded by ESR1 and ESR2 genes, respectively. Objective: The study's aim was to describe a family in which 3 members presented with estrogen insensitivity. Design and Setting: Clinical evaluation and genetic and mutational analysis were performed in an academic medical center. Patients and Interventions: An ESR1 mutation was identified in 2 sisters and 1 brother, originating from a consanguineous Algerian family, who did not enter puberty and presented with delayed bone maturation consistent with estrogen insensitivity. The 2 sisters had enlarged multicystic ovaries. Hormonal evaluation as well as genetic and mutational analysis were performed. Results: Hormonal evaluation revealed extremely high plasma 17ß-estradiol (>50-fold normal range) associated with elevated gonadotropin levels (greater than threefold normal range), highly suggestive of estrogen resistance. The 3 affected patients carried a homozygous mutation of a highly conserved arginine 394 for which histidine was substituted through an autosomal recessive mode of transmission. Structural and functional analysis of the mutant ERα revealed strongly reduced transcriptional activity and the inability to securely anchor the activating hormone, estradiol, compared with wild-type ERα. A group of other potential ER activating ligands were tested, but none overcame the estrogen insensitivity in these patients. Conclusion: Description and analysis of this family of patients with mutant ERα provide additional clinical findings toward identification and characterization of what was previously thought to be a highly rare clinical condition.

NNT mutations: a cause of primary adrenal insufficiency, oxidative stress and extra-adrenal defects.

OBJECTIVE: Nicotinamide nucleotide transhydrogenase (NNT), one of the several genes recently discovered in familial glucocorticoid deficiencies (FGD), is involved in reactive oxygen species detoxification, suggesting that extra-adrenal manifestations may occur, due to the sensitivity to oxidative stress of other organs rich in mitochondria. Here, we sought to identify NNT mutations in a large cohort of patients with primary congenital adrenal insufficiency without molecular etiology and evaluate the degree of adrenal insufficiency and onset of extra-adrenal damages. METHODS: Sanger or massive parallel sequencing of NNT and patient monitoring. RESULTS: Homozygous or compound heterozygous NNT mutations occurred frequently (26%, 13 unrelated families, 18 patients) in our cohort. Seven new mutations were identified: p.Met337Val, p.Ala863Glu, c.3G>A (p.Met1?), p.Arg129*, p.Arg379*, p.Val665Profs*29 and p.Ala704Serfs*19. The most frequent mutation, p.Arg129*, was found recurrently in patients from Algeria. Most patients were diagnosed belatedly (8-18 months) after presenting severe hypoglycemia; others experiencing stress conditions were diagnosed earlier. Five patients also had mineralocorticoid deficiency at onset. One patient had congenital hypothyroidism and two cryptorchidism. In follow-up, we noticed gonadotropic and genitalia impairments (precocious puberty, testicular inclusions, interstitial Leydig cell adenoma, azoospermia), hypothyroidism and hypertrophic cardiomyopathy. Intrafamilial phenotype heterogeneity was also observed. CONCLUSIONS: NNT should be sequenced, not only in FGD, but also in all primary adrenal insufficiencies for which the most frequent etiologies have been ruled out. As NNT is involved in oxidative stress, careful follow-up is needed to evaluate mineralocorticoid biosynthesis extent, and gonadal, heart and thyroid function.

GH deficiency with central precocious puberty: a new rare disorder associated with a developmental defect of the hypothalamic-pituitary area.

CONTEXT: GH deficiency (GHD) associated with central precocious puberty (CPP) has been widely reported in cases of arachnoid cyst, septo-optic dysplasia, brain tumors, or after cerebral radiation therapy. However, idiopathic GHD associated with CPP has been reported in only one isolated case. OBJECTIVE: To evaluate the occurrence and clinical features of the association of nonacquired GHD and CPP. DESIGN AND SETTING: This was a retrospective multicenter study. PATIENTS AND METHODS: The study population was identified through a French nationwide multicenter network (about 3000 patients). We reviewed the medical records of all subjects diagnosed with nonacquired GHD and CPP, with or without developmental abnormalities of the hypothalamic-pituitary axis on cerebral magnetic resonance imaging (MRI), and without any known associated anomaly. RESULTS: We identified four patients with either isolated GHD (n=1) or multiple anterior pituitary hormone deficiencies (n=3). Clinical signs of CPP occurred at 6.4 +/- 2.3 years in boys and 7.5 +/- 0.5 years in girls, and GnRH analog therapy was started at 4.2 +/- 1.6 years after the initiation of recombinant human GH treatment. Cerebral MRI demonstrated ectopic neurohypophysis associated with anterior pituitary hypoplasia in three out of the four patients. The morphology and position of the anterior pituitary and neurohypophysis were normal in one patient who displayed a persistence of the craniopharyngeal canal. CONCLUSIONS: CPP is very rare in patients with nonacquired GHD and is mostly associated with developmental defects in the hypothalamic-pituitary area. Whether molecular mechanisms governing development and activation of the hypothalamic-pituitary axis share dependent factors remains to be explored.