Genetic study of transthyretin amyloid neuropathies: carrier risks among French and Portuguese families.
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Adolescent↗
Biomedical subjects
M Misrahi
Publications and source records attributed to M Misrahi.
Delayed puberty and primary amenorrhea associated with a novel mutation of the human follicle-stimulating hormone receptor: clinical, histological, and molecular studies.
Inactivating mutations of the FSH receptor have been described in rare cases of premature ovarian failure. Only one mutation was associated with a complete phenotype, including delayed puberty, primary amenorrhea, and small ovaries. We describe here a new patient presenting a similar complete phenotype of premature ovarian failure, with high plasma FSH levels associated with very low estrogen and inhibin B levels. No biological response to high doses of recombinant FSH was detected. A novel homozygous Pro(519)Thr mutation was found in this patient. This mutation is located in the second extracellular loop of the FSH receptor, within a motif highly conserved in gonadotropin and TSH receptors. The mutation totally impairs adenylate cyclase stimulation in vitro. FSH binding experiments and confocal microscopy showed that this mutation alters the cell surface targeting of the mutated receptor, which remains trapped intracellularly. Histological studies of the ovaries of the patient showed an increase in the density of small follicles compared with age-matched normal women. A complete block in follicular maturation after the primary stage was also observed. Immunocytochemical studies allowed detection of the expression of c-Kit and proliferation cellular nuclear antigen, whereas no apoptosis was shown by the 3'-end-labeling method. This observation supports the concept that in humans FSH seems mandatory for the initiation of follicular growth only after the primary stage. In our patient complete FSH resistance yields infertility, which is remarkably associated with the persistence of a high number of small follicles.
Adult↗
[Primary ovarian failure and precocious menopause: genetic causes].
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Animals↗
Expression of the thyroid-stimulating hormone receptor in the folliculo-stellate cells of the human anterior pituitary.
TSH secretion from the anterior pituitary is mainly regulated by TRH and thyroid hormones. We hypothesized that in addition the pituitary itself could modulate TSH production by sensing its own TSH release, enabling fine-tuning of TSH secretion. For such an ultra-short loop control, the pituitary should contain a TSH receptor (TSH-R). To find evidence for this we screened a human pituitary complementary DNA library with a digoxigenin-labeled TSH-R probe and found 2 positive clones of 32,000 plaques. One clone was sequenced and found to be completely identical to the thyroid TSH-R. Further proof was obtained by RT-PCR on a human anterior pituitary obtained at autopsy. In situ hybridization and immunohistochemistry confirmed the presence of TSH-R in the anterior pituitary at the messenger ribonucleic acid level as well as the protein level. Moreover, double labeling experiments revealed that TSH-R messenger ribonucleic acid as well as TSH-R protein colocalize with major histocompatibility complex class II expression of folliculo-stellate cells. We conclude that TSH-R is expressed in a subpopulation of folliculo-stellate cells in the human anterior pituitary. This finding suggests ultra-short loop regulation of TSH secretion. Putative recognition of the pituitary TSH-R by TSH-R antibodies might have clinical relevance in Graves' disease.
Adult↗
CCR2B-64I chemokine receptor allele and mother-to-child HIV-1 transmission or disease progression in children. French pediatric HIV infection study group.
The beneficial role of a variant of the chemokine receptor CCR2B (CCR2B-641) in the evolution of HIV-1 infection in adults is still controversial. Furthermore, no studies have been performed in HIV-1-infected children. A multicenter and prospective study of 745 infants born to HIV-1-seropositive mothers was performed. The CCR2B-641 allele was studied in 525 non-African children among whom 523 had been previously genotyped for the CCR5delta32 allele and 220 African children. Of the 745 total, 376 children were infected and 369 were uninfected. In the complete population studied, the children homozygous for the CCR2B-64I allele and the heterozygous children were found distributed equally in the infected (respectively, 1.6% and 21%) and uninfected (respectively, 1.9% and 26.3%) groups (p < .22). Among 376 infected children, the incidence of stage C symptoms (U.S. Centers for Disease Control and Prevention [CDC] classification) or the progression of severe immune deficiency (CD4 <15%, CDC stage 3) was not significantly different in heterozygous infected children or children homozygous for the normal allele (p < .17 and p < .75, respectively). The same lack of protective effect was obtained when a separate analysis was performed in the non-African and African HIV-1-infected children.
Adult↗
Sequential cleavage and excision of a segment of the thyrotropin receptor ectodomain.
The thyrotropin (TSH) receptor belongs to a subfamily of G protein-coupled receptors, which also includes luteinizing hormone and follicle-stimulating hormone receptors. The TSH receptor (TSHR) differs from the latter by the presence of an additional specific segment in the C-terminal part of its ectodomain. We show here that this insertion is excised in the majority of receptor molecules. Preparation of specific monoclonal antibodies to this region, microsequencing, enzyme-linked immunosorbent assay, and immunoblot studies have provided insight into the mechanisms of this excision. In the human thyroid gland, N termini of the transmembrane receptor beta subunit were found to be phenylalanine 366 and leucines 370 and 378. In transfected L cells a variety of other more proximal N termini were found, probably corresponding to incomplete excisions. The most extreme N terminus was observed to lie at Ser-314. These observations suggest that after initial cleavage at Ser-314 the inserted fragment of TSHR is progressively clipped out by a series of cleavage reactions progressing up to amino acids 366-378. The impossibility of recovering the excised fragment from purified receptor, cell membranes, or culture medium supports this interpretation. The cleavage enzyme has previously been shown to be inhibited by BB-2116, an inhibitor of matrix metalloproteases. However, we show here that it is unaffected by tissue inhibitors of metalloproteases. The cleavage enzyme is very similar to TACE (tumor necrosis factor alpha-converting enzyme) in both these characteristics. However, incubation of the TSH receptor with the purified recombinant catalytic domain of TACE, co-transfection of cells with TACE and TSHR expression vectors, and the use of mutated Chinese hamster ovary cells in which TACE is inactive suggested that the TSHR cleavage enzyme is different from TACE. TACE and TSHR cleavage enzyme may thus possibly be related but different members of the adamalysin family of metzincin metalloproteases.
Animals↗
Luteinizing hormone/human chorionic gonadotrophin receptors in various epidermal structures.
Two different monoclonal antibodies recognizing different epitopes were used to study the localization of luteinizing hormone/human chorionic gonadotrophin (LH/hCG) receptors in human skin. Immunolabelling was observed only in the epidermis and derived structures but not in the dermis. The basal, spinal and granular layers were stained, whereas no receptors were detected in the non-nucleated horny cells. In the growing (anagen) hair, immunostaining was found in the inner root sheath below the level of the sebaceous glands and in the outer root sheath above this level. In the resting (telogen) hair, only the latter staining was observed. In the sebaceous glands, only the thin cells close to the walls of the ducts were immunolabelled. In the eccrine sweat glands, the external clear cells were stained in the secretory portion of the gland, whereas only the cells close to the lumen were labelled in the ducts. The distribution of LH/hCG receptors was compared with that of steroidogenic enzymes (side chain cleavage cytochrome P450, adrenodoxin, 3-beta-hydroxy-5-ene steroid dehydrogenase Delta5-Delta4 isomerase, 17-hydroxylase cytochrome P450 and cytochrome P450 aromatase). Only partial overlaps were observed. The presence of LH receptor mRNA in the skin was confirmed by reverse transcription-polymerase chain reaction. Monoclonal antibodies raised against the human follicle-stimulating hormone receptor failed to detect the latter in the epidermal structures and in the dermis. The role of LH and hCG in skin modifications occurring during pregnancy and after the menopause is unknown. These hormones may possibly act by regulating steroidogenic enzymes or by modulating cell growth and differentiation.
Adult↗
Autoantibodies interacting with purified native thyrotropin receptor.
Native thyrotropin receptor (TSHR) was purified by immunoaffinity chromatography from membrane extracts of stably transfected L cells. An ELISA test was devised to study anti-TSHR autoantibodies directly. Comparison of native TSHR with bacterially expressed, denatured TSHR showed that the latter was not recognized by the autoantibodies, suggesting that they bind to conformational epitopes only present on the native receptor. The use of deglycosylated TSHR and of purified receptor ectodomain (alpha-subunit) showed that the autoantibodies recognized only the protein backbone moiety of the receptor and that their epitopes were localized entirely in its ectodomain. Autoantibodies were detected in 45 of 48 subjects with untreated Graves' disease and in 26 of 47 healthy volunteers. The affinity for the receptor was similar in the two groups (Kd = 0.25-1 x 10-10 M) and the autoantibodies belonged to the IgG class in all cases. Although the concentration of autoantibodies was higher in Graves' disease patients (3.50 +/- 0.36 mg.L-1) than in control subjects (1.76 +/- 0.21) (mean +/- SEM), there was an overlap between the groups. Receptor-stimulating autoantibodies (TSAb) were studied by measuring cAMP synthesis in stably transfected HEK 293 cells. Their characteristics (recognition of alpha-subunit, of deglycosylated TSHR, nonrecognition of bacterially expressed denatured receptor) were similar to those of the antibodies detected by the ELISA test. TSAb were only found in individuals with Graves' disease. The ELISA test measures total anti-TSHR antibodies, whereas the test using adenylate cyclase stimulation measures antibodies that recognize specific epitopes involved in receptor activation. Our observations thus disprove the hypothesis according to which Graves' disease is related to the appearance of anti-TSHR antibodies not present in normal subjects. Actually, anti-TSHR antibodies exist in many euthyroid subjects, in some cases even at concentrations higher than those found in patients with Graves' disease. What distinguishes the latter from normal subjects is the existence of subpopulation(s) of antibodies directed against specific epitope(s) of the receptor involved in its activation.
Animals↗
CCR5 delta32 deletion and reduced risk of toxoplasmosis in persons infected with human immunodeficiency virus type 1. The SEROCO-HEMOCO-SEROGEST Study Groups.
This study attempted to determine whether the CCR5 Delta32 deletion affected progression to certain first AIDS-defining illnesses in human immunodeficiency virus type 1-infected patients enrolled in the French SEROCO/HEMOCO/SEROGEST cohorts. Toxoplasmosis onset as a first AIDS-defining illness was significantly delayed in 253 heterozygous patients, compared with 1404 wild type patients. The relative risk of toxoplasmosis associated with heterozygosity was 0. 39 (95% confidence interval, 0.16-0.96) after adjustment for age, CD4 cell count, and primary specific prophylaxis. A nonsignificant protective trend was observed with regard to the onset of mycobacterial, cytomegalovirus, and herpesvirus diseases, but these events were less frequent than toxoplasmosis. Progression to other conditions (e.g., wasting, non-Hodgkin's lymphoma, Kaposi's sarcoma) was similar in the 2 groups as was the frequency of toxoplasmosis as a subsequent AIDS-defining illness. As chemokines are involved in numerous infectious processes, the Delta32 deletion could delay progression to certain opportunistic infections such as toxoplasmosis.
AIDS-Related Opportunistic Infections↗
The same molecular defects of the gonadotropin-releasing hormone receptor determine a variable degree of hypogonadism in affected kindred.
Detailed endocrinological studies were performed in the three affected kindred of a family carrying mutations of the GnRH receptor gene. All three were compound heterozygotes carrying on one allele the Arg262Gln mutation and on the other allele two mutations (Gln106Arg and Ser217Arg). When expressed in heterologous cells, both Gln106Arg and Ser217Arg mutations altered hormone binding, whereas the Arg262Gln mutation altered activation of phospholipase C. The propositus, a 30-yr-old man, displayed complete idiopathic hypogonadotropic hypogonadism with extremely low plasma levels of gonadotropins, absence of pulsatility of endogenous LH and alpha-subunit, absence of response to GnRH and GnRH agonist (triptorelin), and absence of effect of pulsatile administration of GnRH. The two sisters, 24 and 18 yr old, of the propositus displayed, on the contrary, only partial idiopathic hypogonadotropic hypogonadism. They both had primary amenorrhea, and the younger sister displayed retarded bone maturation and uterus development, but both sisters had normal breast development. Gonadotropin concentrations were normal or low, but in both cases were restored to normal levels by a single injection of GnRH. In the two sisters, there were no spontaneous pulses of LH, but pulsatile administration of GnRH provoked a pulsatile secretion of LH in the younger sister. The same mutations of the GnRH receptor gene may thus determine different degrees of alteration of gonadotropin function in affected kindred of the same family.
Adult↗
New natural inactivating mutations of the follicle-stimulating hormone receptor: correlations between receptor function and phenotype.
Premature ovarian failure occurs in almost 1% of women under age 40. Molecular alterations of the FSH receptor (FSHR) have recently been described. A first homozygous mutation of the FSHR was identified in Finland. More recently, we described two new mutations of the FSHR in a woman presenting a partial FSH-resistance syndrome (patient 1). We now report new molecular alterations of the FSHR in another woman (patient 2) who presented at the age of 19 with primary amenorrhea contrasting with normal pubertal development. She had high plasma FSH, and numerous ovarian follicles up to 3 mm in size were evidenced by ultrasonography. Histological and immunohistochemical examination of ovarian biopsies revealed the presence of a normal follicular development up to the antral stage and disruption at further stages. DNA sequencing showed two heterozygous mutations: Asp224Val in the extracellular domain and Leu601Val in the third extracellular loop of FSHR. Cells transfected with expression vectors encoding the wild type or the mutated Leu601Val receptors bound hormone with similar affinity, whereas binding was barely detectable with the Asp224Val mutant. Confocal microscopy showed the latter to have an impaired targeting to the cell membrane. This was confirmed by its accumulation as a mannose-rich precursor. Adenylate cyclase stimulation by FSH of the Leu601Val mutant receptor showed a 12+/-3% residual activity, whereas in patient 1 a 24+/-4% residual activity was detected for the Arg573Cys mutant receptor. These results are in keeping with the fact that estradiol and inhibin B levels were higher in patient 1 and that stimulation with recombinant FSH did not increase follicular size, estradiol, or inhibin B levels in patient 2 in contrast to what was observed for patient 1. Thus, differences in the residual activity of mutated FSHR led to differences in the clinical, biological, and histological phenotypes of the patient.
Adenylyl Cyclases↗
[Mechanisms of polarized targeting of the FSH receptor].
Gonadotropin and TSH receptors belong to a subgroup of G protein-coupled receptors. TSH and FSH receptor present a particular intracellular traffic: they present a polarized basolateral expression in thyroid follicular cells and in Sertoli cells respectively. By contrast, the LH receptor is expressed circumferentially in target gonadic cells. We expressed these receptors in MDCK cells (a well characterized model of polarized epithelial cells) to understand this difference of properties. We show that the three receptors have a polarized basolateral expression in these cells. All contain a basolateral targeting signal. Furthermore, gonadotropin receptors undergo a partial transcytosis which is not observed for the TSH receptor. We show that heterotrimeric G proteins play a role in this mechanism of transcytosis. This effect is not mediated by adenylate cyclase activation and involves a population of G proteins different from that involved in signal transduction. We thus used in vitro mutagenesis to delineate the basolateral localization signal of the FSH receptor. Surprisingly, the signal is localized in the C-terminal tail of the intracellular domain which is not conserved between the three receptors. It contains 14 amino-acids and its activity is mainly dependent on a tyrosine and a leucine residue. The basolateral localization signal of the FSHR is not colinear with its internalization signal. This signal is autonomous and dominant because, when transferred to an apically targeted membrane protein, the neurotrophin receptor, it redirects the chimeric construct to the basolateral domain of MDCK cells. The basolateral localization signal of the FSH receptor is thus the first signal identified for a G protein-coupled receptor and more generally for a hormone receptor.
Animals↗
Gonadotropin receptors.
Gonadotropin receptors belong to a subgroup of G-protein coupled receptors characterized by a large extracellular domain responsible for the binding of the hormone. Soluble, hormone-binding, alternative splicing variants of the LH receptor, are present in high concentration. A mannose rich precursor form of LH and FSH receptor is accumulated inside target cells. FSH receptors are addressed to the basolateral domain of cells through specific signaling mechanisms. Gonadotropin receptors are also present in endothelial cells of target organ vessels and are involved in hormone transcytosis. Various genetic abnormalities of these receptors (and of the GnRH receptor) are discussed.
Animals↗
Loss of function mutations of the GnRH receptor: a new cause of hypogonadotropic hypogonadism.
The association of hypogonadotropic hypogonadism with anosmia defines Kallmann's syndrome. The gene of the X-linked form of this syndrome has been cloned and several mutations described. However, the relatively small number of hypogonadotropic hypogonadic patients with Kallmann's gene defects supports the hypothesis that other genes may be involved. Idiopathic hypogonadotropic hypogonadism (IHH) is not associated with anosmia. The GnRH gene was excluded as a candidate gene in IHH since no abnormality was found in several patients. The action of the GnRH is mediated through a G-protein coupled receptor present in the cell membrane of gonadotropes. The GnRH receptor was thus another candidate gene. Recently, we described the first patient with partial hypogonadotropic hypogonadism without anosmia caused by loss of function mutations of the GnRH receptor. We compare this first family with a new family presenting complete hypogonadotropic hypogonadism and a variable degree of gonadotrope deficiency in the affected kindred, and discuss genotype-phenotype correlation.
Family Health↗
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.
Adult↗
The basolateral localization signal of the follicle-stimulating hormone receptor.
The follicle-stimulating hormone receptor (FSHR) is physiologically localized in the basolateral compartment of the membrane of Sertoli cells. This localization is also observed when the receptor is experimentally expressed in Madin-Darby canine kidney cells. We thus used in vitro mutagenesis and transfection into these polarized cells to delineate the basolateral localization signal of the receptor. The signal was localized in the C-terminal tail of the intracellular domain (amino acids 678-691) at a marked distance of the membrane. Mutation of individual amino acids highlighted the importance of Tyr684 and Leu689. The 14-amino acid sequence was grafted onto the p75 neurotrophin receptor and redirected this apical protein to the basolateral cell membrane compartment. Deletion of amino acids 677-695 did not modify the internalization of the FSHR, showing that the basolateral localization signal of the FSHR is not colinear with its internalization signal.
Amino Acid Sequence↗
Oncogenic potential of a mutant human thyrotropin receptor expressed in FRTL-5 cells.
An abnormal stimulation of the cAMP pathway has been recognized as the primary event in various pathological situations that lead to goitrogenesis or thyroid tumors. Thyroid adenomas are monoclonal neoplasms that become independent of thyroid stimulating hormone (TSH) in their secretory function and growth. Mutated forms of the TSH receptor (TSHR) and the adenylyl cyclase-activating Gs alpha protein, which confer a constitutive activity on these proteins, have been observed in human adenomas. The FRTL-5 rat thyroid cell line is a permanent but untransformed line; the growth of which depends on the presence of TSH, and at least in part, on the stimulation of the cAMP pathway. In order to compare the oncogenic potential of the activated mutant Gs alpha protein and the constitutively activated TSHR, we have transfected FRTL-5 cells with an expression vector bearing either the cDNA of the Gs alpha gene carrying the A201S mutation or the cDNA of the TSH receptor carrying the M453T mutation recently identified in a case of congenital hyperthyroidism. The expression of these two cDNAs was driven by the bovine thyroglobulin gene promoter. We show that, although the expression of both the Gs alpha or TSHR mutant proteins leads to TSH-independent proliferation and to constitutive cAMP accumulation in FRTL-5 cells, only the mutant TSHR is able to induce neoplastic transformation, as demonstrated by growth in semi-solid medium and tumorigenesis in nude mice.
Animals↗