Philippe Touraine

A NOVEL PHENOTYPE RELATED TO PARTIAL LOSS OF FUNCTION MUTATIONS OF THE FOLLICLE STIMULATING HORMONE RECEPTOR

A single natural loss of function mutation of the follicle stimulating hormone receptor (FSHR) has been described to date. Present in the Finnish population it markedly impairs receptor function, blocking follicle development at the primary stage and presenting as primary amenorrhea with atrophic ovaries. When Western European women with this phenotype were examined for FSHR mutations the result was negative, suggesting that other etiologies corresponding to this clinical pattern are markedly more frequent. We now describe a novel phenotype related to mutations provoking a partial loss of function of the FSHR. A woman with secondary amenorrhea had very high plasma gonadotropin concentrations (especially FSH), contrasting with normal sized ovaries and antral follicles up to 5 mm at ultrasonography. Histological and immunohistochemical examination of the ovaries showed normal follicular development up to the small antral stage and a disruption at further stages. The patient was found to carry compound heterozygotic mutations of the FSHR gene: Ile160Thr and Arg573Cys substitutions located, respectively, in the extracellular domain and in the third intracellular loop of the receptor. The mutated receptors, when expressed in COS-7 cells, showed partial functional impairment, consistent with the clinical and histological observations: the first mutation impaired cell surface expression and the second altered signal transduction of the receptor. This observation suggests that a limited FSH effect is sufficient to promote follicular growth up to the small antral stage. Further development necessitates strong FSH stimulation. The contrast between very high FSH levels and normal sized ovaries with antral follicles may thus be characteristic of such patients.

Clinical and Molecular Characterization of a Cohort of 161 Unrelated Women with Nonclassical Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency and 330 Family Members

CONTEXT Nonclassical congenital adrenal hyperplasia (NC-CAH) due to partial 21-hydroxylase deficiency is one of the most frequent autosomal recessive diseases. OBJECTIVE The aim of this study was to determine the genotype/phenotype relationship in probands and family members. PATIENTS AND METHODS A total of 161 NC-CAH unrelated women diagnosed on late-onset symptoms, mainly hirsutism, and post-ACTH 17-hydroxyprogesterone more than 10 ng/ml, and 330 of their relatives was explored. CYP21A2 was genotyped in 124 probands. RESULTS The most frequent mutation was V281L. One severe mutation was found in 63.7% of probands, and surprisingly two severe mutations in four probands. Contrasting with the absence of clinical differences, basal testosterone, and androstenedione, basal and post-ACTH 17-hydroxyprogesterone were significantly higher in probands carrying at least one severe mutation than in those with two mild mutations (P < 0.01). Among the 330 family members, 51 were homozygotes or compound heterozygotes, and 42 were clinically asymptomatic; 242 were heterozygotes and 37 unaffected. Post-ACTH 21-deoxycortisol (21dF) was significantly higher in heterozygotes than in unaffected, however, an overlap existed. In 12 heterozygotes, post-ACTH 21dF was below 0.55 ng/ml, the cutoff value usually accepted for suggesting heterozygosity. CONCLUSIONS The study of family members underlines the variable expression of NC-CAH even within a family, suggesting that modifier factors may modulate phenotype expression. Post-ACTH 21dF cannot reliably detect heterozygous subjects. Considering the high frequency of heterozygotes in the general population, it is essential to genotype the partner(s) of the patients with one severe mutation to offer genetic counseling.

New concepts in prolactin biology

Human prolactin (PRL) is currently viewed as a hormone of pituitary origin, whose production (i.e. serum levels) is controlled by dopamine, whose biological actions relate exclusively to lactation and reproductive functions, for which any genetic disorder is yet to be identified, and whose unique associated pathology is hyperprolactinemia. Both experimental studies and human sample/cohort-based investigations performed during the past decade have considerably widened our perception of PRL biology: i) there are now strong epidemiological arguments supporting the fact that circulating PRL is a risk factor for breast cancer, ii) in addition to the endocrine hormone, locally produced PRL has been documented in several human tissues; there is increasing evidence supporting the tumor growth potency of local PRL, acting via autocrine/paracrine mechanisms, in both rodent models, and human breast and prostate tumors, iii) the first functional germinal polymorphisms of the PRL receptor were recently identified in patients presenting with breast tumors, which involve single amino acid substitution variants exhibiting constitutive activity, iv) human PRL analogs have been engineered, which were shown in experimental models to down-regulate the effects triggered by local PRL (competitive antagonism) or by the constitutively active receptor variants (inverse agonism). The aim of this review is to discuss these novel concepts in PRL biology, including their potential pathophysiological outcomes.

Human prolactin (hPRL) antagonists inhibit hPRL-activated signaling pathways involved in breast cancer cell proliferation.

The involvement of human prolactin (hPRL) in breast cancer has been recently reconsidered based on its autocrine/paracrine proliferative effect described in human mammary tumor epithelial cells. Therefore, there is growing interest in the development of potent hPRL antagonists that may inhibit this effect. We previously designed hPRL analogs displaying antagonistic properties in a human transcriptional bioassay. We now report that the most potent of those analogs, G129R-hPRL, antagonizes all hPRL-induced effects analysed in various breast cancer cell lines, including cell proliferation. The analog per se lacks intrinsic agonistic activity on PRL receptor-activated signaling cascades, cell proliferation and apoptosis, indicating that its mode of action only occurs through competitive inhibition of hPRL. We provide some molecular basis of this antagonistic effect by demonstrating that G129R-hPRL competitively inhibits hPRL-activation of the JAK-STAT and MAPK pathways, two signaling cascades involved in the mitogenic effect of hPRL in mammary epithelial cells. This competitive inhibition persists for at least 48 h, as evidenced by long term analysis of STAT5b activation or of progression through cell cycle. These results are the first demonstration at the molecular level that hPRL antagonists interfering with receptor dimerization disrupt signaling events in breast cancer cells, which prevents hPRL-induced cell proliferation.

Fertility in Women with Nonclassical Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency

OBJECTIVE In contrast to subfertility often reported in women suffering from the classical form of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency, fertility in nonclassical CAH (NC-CAH) has been rarely studied. Our objective was to evaluate fertility in NC-CAH women. MATERIAL AND METHODS We studied 190 NC-CAH women (161 probands + 29 first degree relatives). Only 20 probands had consulted for infertility (12%), either alone or associated with hirsutism or menstrual cycle disorders. The diagnosis was established on post-ACTH 17-hydroxyprogesterone 10 ng/ml or greater and further characterized by CYP21A2 gene analysis. RESULTS Ninety-five of the 190 women wanted pregnancy (aged 26.7 +/- 8.9 yr); 187 pregnancies occurred in 85 women, which resulted in 141 births in 82 of them. Ninety-nine pregnancies (52.9%) occurred before the diagnosis of NC-CAH (96 spontaneously and three with ovulation inducers) whereas 98 occurred after diagnosis (11 spontaneously and 77 with hydrocortisone treatment); 83% of pregnancies were obtained within 1 yr. The rate of miscarriages was 6.5% for pregnancies obtained with glucocorticoid treatment vs. 26.3% without. Two of the 141 infants (1.5%) were born with classical CAH. CONCLUSION Subfertility is mild in NC-CAH. However, the rate of miscarriages is lower in pregnancies occurring with glucocorticoid treatment and argues for treating NC-CAH women wanting pregnancy. In addition, considering the high rate of heterozygotes for CYP21A2 mutations in the general population, it is essential to genotype the partner of patients with a severe mutation to predict the risk of classical CAH and offer genetic counseling.

Genetic investigation of four meiotic genes in women with premature ovarian failure

OBJECTIVE The goal of this study was to determine whether mutations of meiotic genes, such as disrupted meiotic cDNA (DMC1), MutS homolog (MSH4), MSH5, and S. cerevisiae homolog (SPO11), were associated with premature ovarian failure (POF). DESIGN Case-control study. METHODS Blood sampling, karyotype, hormonal dosage, ultrasound, and ovarian biopsy were carried out on most patients. However, the main outcome measure was the sequencing of genomic DNA from peripheral blood samples of 41 women with POF and 36 fertile women (controls). RESULTS A single heterozygous missense mutation, substitution of a cytosine residue with thymidine in exon 2 of MSH5, was found in two Caucasian women in whom POF developed at 18 and 36 years of age. This mutation resulted in replacement of a non-polar amino acid (proline) with a polar amino acid (serine) at position 29 (P29S). Neither 36 control women nor 39 other patients with POF possessed this genetic perturbation. Another POF patient of African origin showed a homozygous nucleotide change in the tenth of DMC1 gene that led to an alteration of the amino acid composition of the protein (M200V). CONCLUSIONS The symptoms of infertility observed in the DMC1 homozygote mutation carrier and in both patients with a heterozygous substitution in exon 2 of the MSH5 gene provide indirect evidence of the role of genes involved in meiotic recombination in the regulation of ovarian function. MSH5 and DMC1 mutations may be one explanation for POF, albeit uncommon.

Long-Term Outcome of Patients with Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency

Aims: Conflicting results exist regarding bone mineral density (BMD), metabolism and reproductive function of adult patients with congenital adrenal hyperplasia (CAH). We evaluated the long-term outcome and the impact of chronic glucocorticoid replacement in these patients. Methods: Physical characteristics, serum hormone concentrations, BMD and metabolism were studied in 45 consecutive CAH adult patients. Results: Among the 36 women, only 14 (39%) had regular menses. Among the 27 women with classical CAH, the mean number of surgical reconstructions of virilized genitalia was 2.1 ± 0.2. Twenty of them (74%) were sexually active. Three men presented with testicular adrenal rest tumors. Twenty-five patients (55%) had decreased BMD at the femoral neck and/or at the lumbar spine. BMI was correlated with the BMD T-score at the femoral neck (p < 0.001) and at the lumbar spine (p < 0.01). Hydrocortisone dose was negatively correlated with the BMD T-score at the femoral neck (p = 0.04). Subjects with osteopenia had a significantly lower BMI and received higher hydrocortisone dose than those with normal BMD. Overweight was found in 21 patients (47%). There was a significantly positive correlation between HOMA and BMI (p < 0.001), and between HOMA and 17-OHP levels (p = 0.016). Conclusions: Adult patients with CAH treated with long-term glucocorticoids are at risk for decreased BMD, increased BMI, and disturbed reproductive function.

Phenotyping and genetic studies of 357 consecutive patients presenting with premature ovarian failure

OBJECTIVE Premature ovarian failure (POF) encompasses a heterogeneous spectrum of conditions, with phenotypic variability among patients. The etiology of POF remains unknown in most cases. We performed a global phenotyping of POF women with the aim of better orienting attempts at an etiological diagnosis. DESIGN AND METHODS We performed a mixed retrospective and prospective study of clinical, biological, histological, morphological, and genetic data relating to 357 consecutive POF patients between 1997 and 2008. The study was conducted at a reproductive endocrinology referral center. RESULTS Seventy-six percent of the patients presented with normal puberty and secondary amenorrhea. Family history was present in 14% of the patients, clinical and/or biological autoimmunity in 14.3%. Fifty-six women had a fluctuating form of POF. The presence of follicles was suggested at ultrasonography in 50% of the patients, and observed in 29% at histology; the negative predictive value of the presence of follicles at ultrasonography was 77%. Bone mineral density alterations were found in 58% of the women. Eight patients had X chromosomal abnormalities other than Turners syndrome, eight other patients evidenced FMR1 pre-mutation. Two other patients had autoimmune polyendocrine syndrome type 2 and 1. CONCLUSION A genetic cause of POF was identified in 25 patients, i.e. 7% of the whole cohort. POF etiology remains most often undiscovered. Novel strategies of POF phenotyping are in such content mandatory to improve the rate of POF patients for whom etiology is identified.

From the molecular biology of prolactin and its receptor to the lessons learned from knockout mice models.

Prolactin (PRL), a polypeptide hormone secreted mainly by the pituitary and, to a lesser extent, by peripheral tissues, affects more physiological processes than all other pituitary hormones combined since it is involved in > 300 separate functions in vertebrates. Its main actions are related to lactation and reproduction. The initial step of PRL action is the binding to a specific membrane receptor, the PRLR, which belongs to the class 1 cytokine receptor superfamily. PRL-binding sites have been identified in a number of tissues and cell types in adult animals. Signal transduction by this receptor is mediated, at least in part, by two families of signaling molecules: Janus tyrosine kinases and signal transducers and activators of transcription (STATs). Disruption of the PRLR gene has provided a new mouse model with which to identify actions directly associated with PRL or any other PRLR ligands, such as placental lactogens. To date, several different phenotypes have been analyzed and are briefly described in this review. Coupled with the SAGE technique, this PRLR knockout model is being used to qualitatively and quantitatively evaluate the expression pattern of hepatic genes in two physiological situations: transcriptomes corresponding to livers from both wild type and PRLR KO mice are being compared, and following statistical analyses, candidate genes presenting a differential profile will be further characterized. Such a new approach will undoubtedly open future avenues of research for PRL targets. To date, no pathology linked to any mutation in the genes encoding PRL or its receptor have been identified. The development of genetic models provides new opportunities to understand how PRL can participate to the development of pathologies throughout life, as for example the initiation and progression of breast cancer.

Molecular Pathology of the FSH receptor: new insights into FSH physiology

Manipulations of mouse genome have helped to elucidate gonadotrophin function but important differences subsist between rodent and human reproduction. Studies of patients with mutations of gonadotrophins or gonadotrophin receptors genes allow understanding their physiological effects in humans. The correlation of the clinical phenotypes of patients with in vitro studies of the mutated receptor residual function and histological and immunohistological studies of the ovarian biopsies permits to understand which stages of follicular development are under FSH control. Total FSH receptor (FSHR) inactivation causes infertility with an early block of follicular maturation remarkably associated with abundant small follicles as in prepubertal ovaries and demonstrates the absolute requirement of FSH for follicular development starting from the primary stage. Partial FSHR inactivation, characterized by normal-sized ovaries, can sustain follicular development up to the early antral stages but incremental levels of FSH stimulation seem to be required for antral follicular growth before selection. These findings contrast with the traditional view of an initial gonadotrophin-independent follicular growth prior to the preantral-early antral stages. The presence of numerous reserve follicles in the ovaries of these patients may permit a future treatment of their infertility. The study of reduced FSHbeta or FSHR activity in genetically modified male mice models and in men suggests a minor impact of the FSHR on masculine fertility. Further studies on patients with a demonstrated total FSHbeta or FSHR inactivation are required to elucidate reported differences in spermatogenesis impairment. Finally, the studies of mutations of gonadotrophins and their receptors demonstrate differences in gonadotrophin function between genetically modified rodents and humans which suggest prudence in extrapolating observations in rodents to human reproduction. Ovarian hyperstimulation syndrome (OHSS) can infrequently arise spontaneously during pregnancy, but most often it is an iatrogenic complication of ovarian stimulation treatments with ovulation drugs for in vitro fertilization. The first genetic cause of familial recurrent spontaneous OHSS was identified as a broadening specificity of the FSHR for hCG due to naturally occurring heterozygous mutations located unexpectedly in the transmembrane domain of the FSHR. Broadening specificity of a G protein-coupled receptor is extremely rare. These observations led to the identification of the etiology of this previously unexplained syndrome and permitted to conceive novel models of FSHR activation. Susceptibility to iatrogenic OHSS or its clinical severity may be associated with FSHR polymorphisms with slightly different activities in vivo as suggested by several studies. The study of larger cohorts is needed to evaluate the clinical impact of these observations in the management of patients undergoing IVF protocols.