Rachel Reynaud

Phenotypical, Biological, and Molecular Heterogeneity of 5α-Reductase Deficiency: An Extensive International Experience of 55 Patients

CONTEXT In 46,XY disorders of sex development, 5α-reductase deficiency is rare and is not usually the first-intention diagnosis in newborn ambiguous genitalia, contrary to partial androgen insensitivity syndrome. Yet the cause of ambiguous genitalia may guide sex assignment, and rapid, precise diagnosis of 5α-reductase deficiency is essential. OBJECTIVE The aim of the study was to describe relevant data for clinical diagnosis, biological investigation, and molecular determination from 55 patients with srd5A2 mutations identified in our laboratory over 20 yr to improve early diagnosis. SETTING The study was performed at Montpellier University Hospital. PATIENTS We studied a cohort of 55 patients with srd5A2 gene mutations. MAIN OUTCOME MEASURE(S) Genetic analysis of srd5A2 was conducted. RESULTS Clitoromegaly (49.1%) and microphallus with various degrees of hypospadias (32.7%) were frequent phenotypes. Female external genitalia (7.3%) and isolated micropenis (3.6%) were rare. Seventy-two percent of patients were initially assigned to female gender; five of them (12.5%) switched to male sex in peripuberty. Over 72% of patients were considered for 5α-reductase deficiency diagnosis when the testosterone/dihydrotestosterone cutoff was 10. In 55 patients (with 20 having a history of consanguinity), we identified 33 different mutations. Five have never been reported: p.G32S, p.Y91H, p.G104E, p.F223S, and c.461delT. Homozygous mutations were present in 69.1% of cases, compound heterozygous mutations in 25.5%, and compound heterozygous mutations alone with the V89L polymorphism in 5.4%. Exons 1 and 4 were most affected, with 35.8 and 21.7% mutant alleles per exon, respectively. CONCLUSIONS In the largest cohort to date, we demonstrate a wide spectrum of phenotypes and biological profiles in patients with 5α-reductase deficiency, whatever their geographical or ethnic origins.

PROKR2 Variants in Multiple Hypopituitarism with Pituitary Stalk Interruption

CONTEXT Pituitary stalk interruption represents a frequent feature of congenital hypopituitarism, but only rare cases have been assigned to a known genetic cause. OBJECTIVE Using a candidate gene approach, we tested several genes as potential causes of hypopituitarism with pituitary stalk interruption. We hypothesized that ectopic posterior pituitary may be a consequence of defective neuronal axon projections along the pituitary stalk or defective angiogenesis of hypophyseal portal circulation. Considering the role of the prokineticin 2 pathway in angiogenesis and neuronal migration, we screened PROK2 and PROKR2 genes. DESIGN PROK2 and PROKR2 and all genes previously known to be involved in hypopituitarism with pituitary stalk interruption (LHX4, HESX1, OTX2, and SOX3) were screened in 72 index cases with pituitary stalk interruption syndrome from the GENHYPOPIT database. In vitro studies were performed to assess the functional consequences of allelic variants. RESULTS We identified two heterozygous PROKR2 mutations (p.Leu173Arg and p.Arg85His) previously reported in isolated hypogonadotroph hypogonadism and a novel PROKR2 variant (p.Ala51Thr) that, in contrast with both other mutations, did not impair receptor signaling activity. Three allelic variants of HESX1 were identified: the heterozygous p.Phe156Ser and the homozygous p.Arg109X mutations were functionally deleterious, whereas p.Ser67Thr was found as a rare allelic variant in association with p.Arg85His PROKR2 mutation in the same patient. CONCLUSIONS We report PROKR2 variants in congenital hypopituitarism with pituitary stalk interruption, suggesting a potential role of the prokineticin pathway in pituitary development.

Pituitary stalk interruption syndrome in 83 patients: novel HESX1 mutation and severe hormonal prognosis in malformative forms

BACKGROUND Pituitary stalk interruption syndrome (PSIS) is a particular entity in the population of patients with hypopituitarism. Only rare cases have a known genetic cause. OBJECTIVES i) To compare subgroups with or without extra-pituitary malformations (EPM) in a cohort of PSIS patients to identify predictive factors of evolution, ii) to determine the incidence of mutations of the known pituitary transcription factor genes in PSIS. Study design We analyzed features of 83 PSIS patients from 80 pedigrees and screened HESX1, LHX4, OTX2, and SOX3 genes. RESULTS PSIS had a male predominance and was rarely familial (5%). Pituitary hypoplasia was observed only in the group with EPM. Multiple hormone deficits were observed significantly more often with versus without EPM (87.5 vs 69.5% respectively). Posterior pituitary location along the stalk was a significant protective factor regarding severity of hormonal phenotype. A novel HESX1 causative mutation was found in a consanguineous family, and two LHX4 mutations were present in familial PSIS. CONCLUSION PSIS patients with EPM had a more severe hormonal disorder and pituitary imaging status, suggesting an antenatal origin. HESX1 or LHX4 mutations accounted for <5% of cases and were found in consanguineous or familial cases.

Is Hypospadias Associated with Prenatal Exposure to Endocrine Disruptors? A French Collaborative Controlled Study of a Cohort of 300 Consecutive Children Without Genetic Defect

BACKGROUND Numerous studies have focused on the association between endocrine-disrupting chemicals (EDCs) and hypospadias. Phenotype variability, the absence of representative comparison groups and concomitant genetic testing prevent any definitive conclusions. OBJECTIVE To identify the role of occupational and environmental exposures to EDCs in nongenetic isolated hypospadias. DESIGN, SETTING, AND PARTICIPANTS A total of 408 consecutive children with isolated hypospadias and 302 normal boys were prospectively included (2009-2014) in a multi-institutional study in the south of France, the area of the country with the highest prevalence of hypospadias surgery. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS In patients without AR, SRD5A2, and MAMLD1 mutations, parental occupational and professional exposures to EDCs were evaluated based on European questionnaire QLK4-1999-01422 and a validated job-exposure matrix for EDCs. Environmental exposure was estimated using the zip code, the type of surrounding hazards, and distance from these hazards. Multivariate analysis was performed. RESULTS Fetal exposure to EDCs around the window of genital differentiation was more frequent in the case of hypospadias (40.00% vs 17.55%, odds ratio 3.13, 95% confidence interval 2.11-4.65). The substances were paints/solvents/adhesives (16.0%), detergents (11.0%), pesticides (9.0%), cosmetics (5.6%), and industrial chemicals (4.0%). Jobs with exposure were more frequent in mothers of hypospadiac boys (19.73% vs 10.26%, p=0.0019), especially cleaners, hairdressers, beauticians, and laboratory workers. Paternal job exposure was more frequent in the cases of hypospadias (40.13% vs 27.48%, p=0.02). Industrial areas, incinerators, and waste areas were more frequent within a 3-km radius for mothers of hypospadiac boys (13.29% vs. 6.64%, p<0.00005). Association of occupational and environmental exposures increases this risk. CONCLUSIONS This multicenter prospective controlled study with a homogeneous cohort of hypospadiac boys without genetic defects strongly suggests that EDCs are a risk factor for hypospadias through occupational and environmental exposure during fetal life. The association of various types of exposures may increase this risk. PATIENT SUMMARY Our multi-institutional study showed that parental professional, occupational, and environmental exposures to chemical products increase the risk of hypospadias in children.

Mutations in the maternally imprinted gene MKRN3 are common in familial central precocious puberty

CONTEXT AND OBJECTIVE Idiopathic central precocious puberty (iCPP) is defined as early activation of the hypothalamic-pituitary-gonadal axis in the absence of identifiable central lesions. Mutations of the makorin RING finger 3 (MKRN3) gene are associated with iCPP. We aimed to assess the frequency of MKRN3 mutations in iCPP and to compare the phenotypes of patients with and without MKRN3 mutations. DESIGN An observational study was carried out on patients recruited at pediatric hospitals in France and Italy. Forty-six index CPP cases were screened for mutations in the MKRN3 coding sequence: 28 index cases of familial cases and 18 cases did not report any familial history of CPP. The endocrine phenotype was compared between MKRN3 mutated and non-mutated patients. RESULTS MKRN3 mutations were identified in one sporadic and 13 familial cases. We identified five new heterozygous missense mutations predicted to be deleterious for protein function and two frameshift mutations, one new and the other recurrent, predicted to result in truncated proteins. Age at puberty onset varied very little among patients with MKRN3 mutations and puberty occurred earlier in these patients than in those without MKRN3 mutations (6.0 years (5.4-6.0) vs 7.0 years (6.0-7.0), P=0.01). CONCLUSIONS MKRN3 mutations are common in familial iCPP. MKRN3 is one of the gatekeepers of the postnatal activation of the gonadotropic axis.

MECHANISMS IN ENDOCRINOLOGY: AN UPDATE IN THE GENETIC AETIOLOGIES OF COMBINED PITUITARY HORMONE DEFICIENCY

Over the last 5 years, new actors involved in the pathogenesis of combined pituitary hormone deficiency in humans have been reported: they included a member of the immunoglobulin superfamily glycoprotein and ciliary G protein-coupled receptors, as well as new transcription factors and signalling molecules. New modes of inheritance for alterations of genes encoding transcription factors have also been described. Finally, actors known to be involved in a very specific phenotype (hypogonadotroph hypogonadism for instance) have been identified in a wider range of phenotypes. These data thus suggest that new mechanisms could explain the low rate of aetiological identification in this heterogeneous group of diseases. Taking into account the fact that several reviews have been published in recent years on classical aetiologies of CPHD such as mutations of POU1F1 or PROP1, we focused the present overview on the data published in the last 5 years, to provide the reader with an updated review on this rapidly evolving field of knowledge.

Minor Hypospadias: The “Tip of the Iceberg” of the Partial Androgen Insensitivity Syndrome

Background Androgens are critical in male external genital development. Alterations in the androgen sensitivity pathway have been identified in severely undermasculinized boys, and mutations of the androgen receptor gene (AR) are usually found in partial or complete androgen insensitivity syndrome (AIS). Objective The aim of this study was to determine whether even the most minor forms of isolated hypospadias are associated with AR mutations and thus whether all types of hypospadias warrant molecular analysis of the AR. Materials and Methods Two hundred and ninety-two Caucasian children presenting with isolated hypospadias without micropenis or cryptorchidism and 345 controls were included prospectively. Mutational analysis of the AR through direct sequencing (exons 1–8) was performed. In silico and luciferase functional assays were performed for unreported variants. Results Five missense mutations of the AR were identified in 9 patients with glandular or penile anterior (n = 5), penile midshaft (n = 2) and penile posterior (n = 2) hypospadias, i.e., 3%: p.Q58L (c.173A>T), 4 cases of p.P392S (c.1174C>T), 2 cases of p.A475V (c.1424C>T), p.D551H (c.1651G>C) and p.Q799E (c.2395C>G). None of these mutations was present in the control group. One mutation has never been reported to date (p.D551H). It was predicted to be damaging based on 6 in silico models, and in vitro functional studies confirmed the lowered transactivation function of the mutated protein. Three mutations have never been reported in patients with genital malformation but only in isolated infertility: p.Q58L, p.P392S, and p.A475V. It is notable that micropenis, a cardinal sign of AIS, was not present in any patient. Conclusion AR mutations may play a role in the cause of isolated hypospadias, even in the most minor forms. Identification of this underlying genetic alteration may be important for proper diagnosis and longer follow-up is necessary to find out if the mutations cause differences in sexual function and fertility later in life.

Genetic causes of combined pituitary hormone deficiencies in humans

Congenital hypopituitarism is a rare disease, usually induced by mutations of genes coding for transcription factors involved in pituitary development. PROP1 mutations represent the first cause of identified congenital hypopituitarism. Current techniques only identify 10-20% of congenital hypopituitarism etiologies, suggesting that new techniques are needed to improve this ratio. This should lead to a better management and follow-up of patients presenting with combined pituitary hormone deficiencies.

Case Report of GNAS Epigenetic Defect Revealed by a Congenital Hypothyroidism

Pseudohypoparathyroidism (PHP) is a group of disorders characterized by end-organ resistance to the parathyroid hormone (PTH). PHP type 1A includes multihormone resistance syndrome, Albright’s hereditary osteodystrophy, and obesity and is caused by mutations in GNAS exon 1 through 13. PHP type 1B (PHP1B), caused by epigenetic changes in the GNAS locus, was initially described as an isolated resistance to PTH. Epigenetic changes in GNAS have also been reported in patients who display mild Albright’s hereditary osteodystrophy or mild thyroid-stimulating hormone (TSH) resistance without mutation of GNAS. Here we report a case of PHP caused by epigenetic changes in GNAS in a patient with congenital hypothyroidism. The patient was referred for a positive newborn screening for hypothyroidism (TSH 50 mIU/L). She exhibited severe clinical features of congenital hypothyroidism. The thyroid was in place, and etiologic explorations were negative. TSH was normalized under L-thyroxin, and the symptoms disappeared, except for a macroglossia. In childhood, PHP was suspected in addition to elevated PTH, obesity, brachydactyly, and a rounded face. Sequencing, methylation analysis, and large deletion research were performed in GNAS. No genetic mutations were found. Methylation analysis revealed a broad epigenetic defect without deletion in GNAS consistent with sporadic PHP1B. The multilocus methylation analysis were negative. This finding expands the known onsets of PHP1B and emphasizes the need for a new PHP classification system. This case report has important consequences for the etiologic diagnosis of congenital hypothyroidism because it adds a new cause of the disease.

Holter glycémique et dépistage du diabète chez l'enfant atteint de mucoviscidose

AIMS In the past few years, survival has increased for people with cystic fibrosis (CF). Diabetes is an important complication of CF caused by pancreatic insufficiency, which reduces insulin secretion. Because of increased longevity of patients with CF, the prevalence of CF-related diabetes (CFRD) has increased. CFRD is associated with increased mortality and morbidity. Several studies have reported a decline in nutritional and pulmonary status 2-4 years before the diagnosis of CFRD. The introduction of insulin treatment can produce clinical improvement in weight and lung function. The oral glucose tolerance test is currently the reference method in screening for CFRD, but the current definition of diabetes based on the 2-h post-load plasma glucose level may not be the most accurate method for early detection of glucose tolerance abnormalities in CF. The continuous glucose monitoring system (CGMS) has been described as a useful tool for early detection of hyperglycemia in the CF patient. We tested the CGMS in CF patients with unexplained alteration of their general status. The aim of this study was to assess the value of the CGMS in this population. METHODS An annual OGTT (following World Health Organization recommendations) was conducted as a screening test to identify CFRD in patients aged over 10 years or patients aged under 10 years with a poorer clinical status. The CGMS was performed in patients with unexplained worsened clinical status and without diabetes in OGTT. RESULTS Forty-two patients aged from 8.5 to 19 years were screened using OGTT for CFRD. According to ADA criteria, 23 patients (54.8%) displayed normal glucose tolerance, 14 (33.3%) impaired glucose tolerance, and 5 diabetes (11.9%). Out of 37 nondiabetic, the CGMS was used in 20 patients with unexplained altered general status. The CGMS revealed peaks of glucose values greater than 2 g/L in 16 patients, 9 patients with normal glucose tolerance, and 7 patients with impaired glucose tolerance. The mean CGMS glucose and time of glycemic monitoring above 1.4 g/L increased in patients with peaks greater than 2 g/L compared to patients without peaks (p=0.0016 and p=0.0069 respectively). After analysis of the CGMS, the prevalence of diabetes increased from 11.9 to 50%. Three patients aged less than 10 years with a normal OGTT profile presented glycemic peaks greater than 2 g/L during CGMS. CONCLUSION CGMS revealed more glucose metabolism abnormalities than OGTT in patients with unexplained altered general status.