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Background Cardiac and neurological involvements are the main clinical features of hereditary transthyretin (ATTRv) amyloidosis. Few data are available about ATTRv amyloid nephropathy (ATTRvN). Methods We retrospectively included 30 patients with biopsy-proven ATTRvN [V30M (26/30) including two domino liver recipients, S77Y (2/30), V122I (1/30) and S50R (1/30) variants] from two French reference centers. We described the pathological features by comparing amyloid deposits distribution to patients with AL or AA amyloidosis, and sought to determine clinicopathological correlation with known disease-modifying factors such as TTR variant, gender and age at diagnosis. Results In comparison with AL and AA amyloidosis, ATTRv patients had similar glomerular, arteriolar and arterial amyloid deposits, but more cortical and medullary tubulointerstitial (33%, 44%, 77%, P = .03) involvement. While the presence of glomerular deposits is associated with the range of proteinuria, some patients with abundant glomerular ATTRv amyloidosis had no significant proteinuria. V30M patients had more glomerular (100% and 25%, odds ratio = 114, 95% confidence interval 3.85–3395.00, P = .001) deposits, and higher estimated glomerular filtration rate [50 (interquartile range 44–82) and 27 (interquartile range 6–31) mL/min/1.73 m², P = .004] than non-V30M patients. We did not find difference in amyloid deposition according to gender or age at diagnosis. Conclusion ATTRvN affects all kidney compartments, but compared with AL/AA amyloidosis, ATTRvN seems to involve more frequently tubulointerstitial areas. V30M patients represents the dominant face of the disease with a higher risk of glomerular/arteriolar involvement. ATTRvN should thus be considered in patients, and potential relatives, with ATTRv amyloidosis and kidney dysfunction, regardless of proteinuria level.

BackgroundPrimary ovarian insufficiency (POI) affects 1% of women under 40 years and is a public health problem. The genetic causes of POI are highly heterogeneous with isolated or syndromic forms. Recently, variants in genes involved in DNA repair have been shown to cause POI. Notably, syndromic POI with Fanconi anaemia (FA) traits related to biallelic BRCA2 truncated variants has been reported. Here, we report a novel phenotype of isolated POI with a BRCA2 variant in a consanguineous Turkish family.MethodsExome sequencing (ES) was performed in the patient. We also performed functional studies, including a homologous recombination (HR) test, cell proliferation, radiation-induced RAD51 foci formation assays and chromosome breakage studies in primary and lymphoblastoid immortalised cells. The expression of BRCA2 in human foetal ovaries was studied.ResultsES identified a homozygous missense c.8524C>T/p.R2842C-BRCA2 variant. BRCA2 defects induce cancer predisposition and FA. Remarkably, neither the patient nor her family exhibited somatic pathologies. The patient’s cells showed intermediate levels of chromosomal breaks, cell proliferation and radiation-induced RAD51 foci formation compared with controls and FA cells. R2842C-BRCA2 only partially complemented HR efficiency compared with wild type-BRCA2. BRCA2 is expressed in human foetal ovaries in pachytene stage oocytes, when meiotic HR occurs.ConclusionWe describe the functional assessment of a homozygous hypomorphic BRCA2 variant in a patient with POI without cancer or FA trait. Our findings extend the phenotype of BRCA2 biallelic alterations to fully isolated POI. This study has a major impact on the management and genetic counselling of patients with POI.

Study question To identify the etiology of isolated Primary Ovarian Insufficiency (POI) in a patient from an Indian consanguineous family. Summary answer A homozygous nonsense mutation of SPIDR in the patient yielded chromosomal instability: first evidence of a role of this gene in DNA repair. What is known already POI, affecting 1% of women under 40, is a public health problem. To date ∼ 70% of cases remain idiopathic. The leap due to exome sequencing, led to the identification of ∼ 80 genes, often in single or few cases. SPIDR was recently identified as a scaffolding protein connecting RAD51, a central player in homologous recombination, to BLM, a helicase implicated in the integrity of the genome. But its precise role is still unknown. A SPIDR mutation was previously associated with POI. However, contradictory conclusions were reported on the mechanism of SPIDR action and on its pathogenic role in POI. Study design, size, duration Prospective genetic study of a cohort of 150 pateints with POI worldwide using a custom-made targeted next generation sequencing (NGS) panel comprising 60 known POI-causing genes. A single patient was found mutated in SPIDR. Cytogenetic studies were performed to analyse the consequences of the mutation on DNA repair and sister chromatide exchanges (SCE). Participants/materials, setting, methods The patient with SPIDR mutation had POI with primary amenorrhea, delayed puberty and streaks ovaries. She was born to consanguineous Indian parents. No other mutation was detected in our cohort of 150 patients with POI. Targeted NGS was performed in the proposita. Familial segregation was performed by Sanger sequencing. Mitomycin C (MMC)-induced chromosomal breakages were studied and a sister chromatid exchange (SCE) assay was performed in patient’s peripheral lymphocytes. Main results and the role of chance We identified a novel homozygous nonsense mutation in the exon 7 of SPIDR (KIAA0146) c.814C>T, R272*, predicted to yield either a truncated protein, or a non-sense-mediated mRNA decay. The patient’s cells display increased chromosomal fragility with high MMC-induced chromosomal breaks when compared to a control. Remarkably, there was no increased SCE. In the previous report of a SPIDR mutation in POI, no cytogenetic studies were performed, and contradictory results were obtained on a homologous recombination test between the two sisters, either enhanced or reduced. In conclusion, we show here that inactivation of SPIDR results in a defect of double strand DNA damage repair, similar to alteration of the RAD51 pathway. There was no increased SCE, the hallmark of the BLM pathway. This observation has major consequences for this patient’s care : indeed mutations of DNA-repair genes may also yield to tumors/cancers. A long follow-up of the patient is needed in a multidisciplinary team to detect possible comorbidities. Indeed, even in the absence of somatic symptomatology, the patient has enhanced chromosomal instability highlighted by cytogenetic studies, that may yield tumor-predisposition. Limitations, reasons for caution No other mutation of SPIDR in the replication cohort of 150 POI patients. SPIDR mutation are thus very rare world-wide. Wider implications of the findings: This is the first evidence of chromosomal instability associated with SPIDR defect, providing strong evidence for a role of SPIDR in double strand DNA damage repair in humans and for its causal role in POI. Our study improves the knowledge on SPIDR function and confirms its involvement in POI worldwide. Trial registration number Not applicable

International audience; Premature ovarian insufficiency (POI) is a rare pathology affecting 1-2% of under-40 year-old women, 1 in 1000 under-30 year-olds and 1 in 10,000 under-20 year-olds. There are multiple etiologies, which can be classified as primary (chromosomal, genetic, auto-immune) and secondary or iatrogenic (surgical, or secondary to chemotherapy and/or radiotherapy). Despite important progress in genetics, more than 60% of cases of primary POI still have no identifiable etiology; these cases are known as idiopathic POI. POI is defined by the association of 1 clinical and 1 biological criterion: primary or secondary amenorrhea or spaniomenorrhea of>4 months with onset before 40 year of age, and elevated follicle-stimulating hormone (FSH)>25IU/L on 2 assays at>4 weeks' interval. Estradiol level is low, and anti-Müllerian hormone (AMH) levels have usually collapsed. Initial etiological work-up comprises auto-immune assessment, karyotype, FMR1 premutation screening and gene-panel study. If all of these are normal, the patient and parents may be offered genome-wide analysis under the "France Génomique" project. The term ovarian insufficiency suggests that the dysfunction is not necessarily definitive. In some cases, ovarian function may fluctuate, and spontaneous pregnancy is possible in around 6% of cases. In confirmed POI, hormone replacement therapy is to be recommended at least up to the physiological menopause age of 51 years. Management in a rare diseases center may be proposed.

Spermatogenesis and folliculogenesis involve cell–cell interactions and gene expression orchestrated by luteinizing hormone (LH) and follicle-stimulating hormone (FSH). FSH regulates the proliferation and maturation of germ cells independently and in combination with LH. In humans, the requirement for high intratesticular testosterone (T) concentration in spermatogenesis remains both a dogma and an enigma, as it greatly exceeds the requirement for androgen receptor (AR) activation. Several data have challenged this dogma. Here we report our findings on a man with mutant LH beta subunit (LHβ) that markedly reduced T production to 1–2% of normal., but despite this minimal LH stimulation, T production by scarce mature Leydig cells was sufficient to initiate and maintain complete spermatogenesis. Also, in the LH receptor (LHR) knockout (LuRKO) mice, low-dose T supplementation was able to maintain spermatogenesis. In addition, in antiandrogen-treated LuRKO mice, devoid of T action, the transgenic expression of a constitutively activating follicle stimulating hormone receptor (FSHR) mutant was able to rescue spermatogenesis and fertility. Based on rodent models, it is believed that gonadotropin-dependent follicular growth begins at the antral stage, but models of FSHR inactivation in women contradict this claim. The complete loss of FSHR function results in the complete early blockage of folliculogenesis at the primary stage, with a high density of follicles of the prepubertal type. These results should prompt the reassessment of the role of gonadotropins in spermatogenesis, folliculogenesis and therapeutic applications in human hypogonadism and infertility.

Endometriosis, the hormone-dependent extrauterine dissemination of endometrial tissue outside the uterus, affects 5–15% of women of reproductive age. Pathogenesis remains poorly understood as well as the estrogen production by endometriotic tissue yielding autocrine growth. Estrogens (E2) are normally produced by the ovaries. We investigated whether aberrant “ovarian-like” differentiation occurred in endometriosis. 69 women, with (n = 38) and without (n = 31) histologically proven endometriosis were recruited. Comparative RT-qPCR was performed on 20 genes in paired eutopic and ectopic lesions, together with immunohistochemistry. Functional studies were performed in primary cultures of epithelial endometriotic cells (EEC). A broaden ovarian-like differentiation was found in half eutopic and all ectopic endometriosis with aberrant expression of transcripts and protein for the transcription factors GATA4 and GATA6 triggering ovarian differentiation, for the FSH receptor (FSHR) and the ovarian hormone INSL3. Like in ovaries the FSHR induced aromatase, the key enzyme in E2 production, and vascular factors in EEC. The LH receptor (LHR) was also aberrantly expressed in a subset of ectopic endometriosis (21%) and induced strongly androgen-synthesizing enzymes and INSL3 in EEC, as in ovaries, as well as endometriotic cell growth. The ERK pathway mediates signaling by both hormones. A positive feedback loop occurred through FSHR and LHR-dependent induction of GATA4/6 in EEC, as in ovaries, enhancing the production of the steroidogenic cascade. This work highlights a novel pathophysiological mechanism with a broadly ovarian pattern of differentiation in half eutopic and all ectopic endometriosis. This study provides new tools that might improve the diagnosis of endometriosis in the future., SCOPUS: ar.j, info:eu-repo/semantics/published

Primary ovarian insufficiency (POI) affects 1% of women under 40 years and is a public health problem. The genetic causes of POI are highly heterogeneous with isolated or syndromic forms. Recently, variants in genes involved in DNA repair have been shown to cause POI. Notably, syndromic POI with Fanconi anaemia (FA) traits related to biallelicExome sequencing (ES) was performed in the patient. We also performed functional studies, including a homologous recombination (HR) test, cell proliferation, radiation-induced RAD51 foci formation assays and chromosome breakage studies in primary and lymphoblastoid immortalised cells. The expression ofES identified a homozygous missense c.8524CT/p.R2842CWe describe the functional assessment of a homozygous hypomorphic

Context Primary Ovarian insufficiency (POI) affects 1% of women aged Objective To identify the cause of a familial POI in a consanguineous Turkish family. Design Exome sequencing was performed in the proposita and her mother. Chromosomal breaks were studied in lymphoblastoid cell lines treated with mitomycin (MMC). Setting and patients The proposita presented intrauterine and postnatal growth retardation, multiple pilomatricomas in childhood, and primary amenorrhea. She was treated with growth hormone (GH) from age 14 to 18 years. Results We identified a novel nonsense variant in exon 9 of the minichromosome maintenance complex component 8 gene (MCM8) NM_001281522.1: c0.925C > T/p.R309* yielding either a truncated protein or nonsense-mediated messenger ribonucleic acid decay. The variant was homozygous in the daughter and heterozygous in the mother. MMC induced DNA breaks and aberrant metaphases in the patient’s lymphoblastoid cells. The mother’s cells had intermediate but significantly higher chromosomal breaks compared with a control. Conclusion We describe a novel phenotype of syndromic POI related to a novel truncating MCM8 variant. We show for the first time that spontaneous tumors (pilomatricomas) are associated with an MCM8 genetic defect, making the screening of this gene necessary before starting GH therapy in patients with POI with short stature, especially in a familial or consanguineous context. Appropriate familial monitoring in the long term is necessary, and fertility preservation should be considered in heterozygous siblings to avoid rapid follicular atresia.

Primary Ovarian insufficiency (POI) affects 1% of women under forty. We studied a patient with a non-syndromic POI, from a consanguineous Turkish family. Exome sequencing identified a homozygous missense variant c.8524C>T/p.R2842C in BRCA2. BRCA2 is a major player in homologous recombination (HR). BRCA2 deficiency induces cancer predisposition and Fanconi Anemia (FA). Remarkably, neither the patient nor her family exhibit somatic pathologies. The patient’s somatic cells presented intermediate levels of chromosomal breaks, cell proliferation and radiation-induced RAD51 foci formation when compared to controls, the heterozygous mother’s and FA cells. R2842C-BRCA2 partially complemented BRCA2 depletion for double-strand break-induced HR. The residual HR function in patient’s cells could explain the absence of somatic pathology. BRCA2 is expressed in human fetal ovaries in pachytene stage oocytes, when meiotic HR occurs. This study has a major impact on the understanding of genome maintenance in somatic and meiotic cells and on the management of POI patients.

Primary ovarian insufficiency (POI) affects ~ 1–3, 7% of women under forty and is a public health problem. Most causes are unknown, but an increasing number of genetic causes have been identified recently. The identification of such causes is essential for genetic and therapeutic counseling in patients and their families. We performed whole exome sequencing in two Caucasian sisters displaying non syndromic POI and their unaffected mother. We identified two novel pathogenic variants in STAG3 encoding a meiosis-specific subunit of the cohesin ring, which ensures correct sister chromatid cohesion: a c.3052delC truncating mutation in exon 28 yielding p.Arg1018Aspfs*14, and a c.659T > G substitution in exon seven yielding p.Leu220Arg. Leu220, highly conserved throughout species, belongs to the STAG domain conserved with other mitotic subunits of the cohesion complex STAG1 and 2. In silico analysis reveals that this substitution markedly impacts the structure of this domain. The truncation removes the last 206 C-terminal residues, not conserved in STAG1 and 2, supporting an important specific role in STAG3, especially meiosis. This is the first occurrence of STAG3 mutations in a Caucasian family. Very little is known about the function of STAG proteins domains. The “knock out-like” phenotype described here supports the crucial role of a single residue in the STAG domain and of the C-terminal region in STAG3 function. In conclusion, this observation shows the necessity to perform the genetic study of POI worldwide including STAG3. This could lead to appropriate genetic counseling and long term follow-up since these patients may develop ovarian tumors.

International audience; Primary ovarian insufficiency (POI) affects similar to 1% of women before 40 years of age. The recent leap in genetic knowledge obtained by next generation sequencing (NGS) together with animal models has further elucidated its molecular pathogenesis, identifying novel genes/pathways. Mutations of > 60 genes emphasize high genetic heterogeneity. Genome-wide association studies have revealed a shared genetic background between POI and reproductive aging. NGS will provide a genetic diagnosis leading to genetic/therapeutic counseling: first, defects in meiosis or DNA repair genes may predispose to tumors; and second, specific gene defects may predict the risk of rapid loss of a persistent ovarian reserve, an important determinant in fertility preservation. Indeed, a recent innovative treatment of POI by in vitro activation of dormant follicles proved to be successful.

Hepatitis C virus (HCV) seroprevalence is highly diverse among human immunodeficiency virus-1 (HIV-1) infected patients, ranging between 10% of HIV-1 infected homo-bisexuel men, to >92% in patients infected with HIV-1 who acquired HIV-1 through intravenous drug use. Thus, being HCV-free while having acquired HIV-1 via intravenous drug use is a rare situation. Claudin-1 is a protein involved in intracellular tight-junctions and has been identified as a major cellular co-receptor for HCV infection. Our objective was to determine whether Claudin-1 gene (CLDN1) mutations might be involved in natural resistance to HCV infection. We conducted a case-control study. All recruited patients acquired HIV-1 infection via intravenous drug use route before 1995. The case study patients remained free from HCV infection (negative anti-HCV antibodies and HCV-RNA). The control study patients was co-infected with HCV (positive anti-HCV antibodies). Direct genomic sequencing of the CLDN1 gene coding region and adjacent intron/exons junctions was performed from peripheral blood mononuclear cells. A total of 138 Caucasian patients were enrolled. Twenty-two patients (cases) were free from HCV infection and 116 (controls) were co-infected with HCV. We found single nucleotide polymorphisms (SNPs) described previously with no significant differences in allele frequencies between cases and controls. In conclusion, despite being a major cellular co-receptor for HCV entry in vitro, we did not identify any specific substitution in CLDN1 gene coding region in our study patients highly exposed but resistant to HCV infection in vivo. Other cellular co-factors involved in HCV infection should be investigated in this highly-exposed intravenous drug users patients. J. Med. Virol. 87:619–624, 2015. © 2015 Wiley Periodicals, Inc.

Premature ovarian insufficiency (POI) affects 1% to 2% of women under 40 years. Bone morphogenetic protein 15 (BMP15) variants have been described in POI. We studied a family with 2 sisters compound heterozygous for deletions in the BMP15 gene on chromosome Xp11.22 yielding a human "knockout-like" effect: a c.151_152delGA deletion yielded a p.Glu51IlefsTer27 mutation transmitted by the hemizygous father and a c.189_198delAGGGCATTCAinsTG deletion/insertion yielded a p.Glu64AlafsTer12 mutation transmitted by the heterozygous mother. Both deletions resulted in frameshifts with premature stop codons at positions 78 and 76 in the proregion, precluding mature BMP15 production. One sister had primary amenorrhea and the other primo-secondary amenorrhea. No bone abnormality was observed. Despite streak ovaries devoid of follicles on ultrasonography, anti-Mullerian hormone (AMH) levels were low but detectable suggesting the presence of growing follicles. Five years later, AMH was undetectable in both sisters, 1 had received an egg donation. BMP15 did not seem critical for follicles to enter the growth phase. Genetic counselling should be performed and fertility preservation discussed before progressive loss of follicular reserve. The fertile heterozygous mother did not support previous reports of BMP15 haploinsufficiency and gene dosage in humans, as in bovine species. The hemizygous brother had asthenozoospermia, consistent with previous observations in bulls with a variant BMP15.

Folliculogenesis requires communication between granulosa cells and oocytes, mediated by connexin-based gap junctions. Connexin 37 (Cx37)-deficient female mice are infertile. The present study assessed Cx37 deficiency in patients with primary ovarian insufficiency (POI). A candidate gene study was performed in patients and controls from the National Genotyping Center (Evry, France) including 58 Caucasian patients with idiopathic isolated POI and 142 Caucasian controls. Direct genomic sequencing of the coding regions of the GJA4 gene (encoding Cx37) was performed with the aim to identify a deleterious variant associated with POI and absent in ethnically matched controls. A single Cx37 variant absent in the control population was identified, namely a c.946G>A heterozygous substitution leading to a p.Gly316Ser variant that was present in two POI patients. This variant was absent in all Caucasian controls from various databases, and has been observed exclusively in African populations. This variant was identified to have a dominant negative effect in HeLa cells in vitro to alter connexon function (by 67.2±7.17%), as determined by Gap-fluorescence recovery after photobleaching. The alteration principally resulted from a decrease of cell surface connexons due to altered trafficking (by 47.73±8.59%). In marked contrast to this observation, a p.Pro258Ser variant frequent in all ethnic populations in databases had no functional effect in vitro. In conclusion, the present study reported on a Cx37 variant in two Caucasian POI patients, which was absent in control Caucasian populations, and which had a deleterious effect in vitro. It is therefore suggested that in the genetic context of the Caucasian population, this variant may contribute to POI.

International audience; Objective To compare the natural history of familial transthyretin amyloid polyneuropathies (FAP) due to the Val30Met, Ser77Tyr, and Ile107Val mutations in France with the classical Portuguese Val30Met FAP. Methods We compared 84 French patients with a control group of 110 Portuguese patients carrying the Val30Met mutation also living in France, all referred to and followed at the French National FAP Reference Center from 1988 to 2010. Clinical examination, functional and walking disability scores, nerve conduction studies, and muscle biopsies are reported. We also conducted a comprehensive literature review to further determine the range of phenotypic expression. Results By comparison with Portuguese Val30Met FAP, French Ile107Val, Ser77Tyr, and LateVal30Met FAP showed more rapid and severe disease progression; onset of gait disorders was 3 times more rapid (p 50 years; LateMet30) FAP (p = 0.0005). Other distinctive features relative to the Portuguese patients included atypical clinical presentations, demyelination on nerve conduction studies (p = 0.0005), and difficult identification of amyloid deposits in nerve and muscle biopsies. Interpretation Ile107Val and LateMet30 mutations are associated with the most debilitating and severe FAP ever described, with rapid onset of tetraparesis and shorter median survival. It could be explained by frequent large‐fiber involvement and associated demyelination and more severe axonal loss. These findings have major implications for genetic counseling and patient management as new therapeutic options are being assessed in clinical trials (TTR gene silencing). Ann Neurol 2015;78:901–916

Discordant expression of Familial Amyloid Neuropathy (FAP) in monozygotic twins is a rare event. Only five such cases have been described in the literature so far. We report the clinical, neurophysiologic and autonomic findings of Brazilian monozygotic twins discordant for the expression of FAP type I. Twin I first presented symptoms at the age of 21, when his brother was completely asymptomatic. Twin 2 only presented symptoms at the age of 25, almost four years after his brother. Both brothers eventually developed the complete phenotype of FAP type I. The occurrence of monozygotic twins discordant for the expression of FAP type I suggests that other factors beside TTR gene mutations should play an important role in the pathogenesis of this condition. Environmental factors, as well as modifier genetic loci are likely to modulate the expression of FAP type I and the study of cases such as the one presented here may help to identify some of these factors.

Resume La maladie de Wilson est une genopathie autosomale recessive se traduisant par une accumulation pathologique de cuivre dans certains tissus (notamment le foie, le cerveau, le rein et la cornee). Le tableau clinique, parfois tres grave, est a predominance hepatique, neurologique et psychiatrique. Le gene en cause, localise sur le chromosome 13, code une proteine de transport du cuivre, l' ATP7B , qui assure son passage dans la voie secretoire ou il est incorpore a la ceruloplasmine et qui intervient dans son excretion biliaire. Les mutations de ce transporteur sont responsables de la maladie. Le diagnostic qui repose sur un faisceau de presomptions clinico-biologiques (baisse de la ceruloplasmine, presence d'un anneau corneen de Kayser-Fleischer, anomalies hepatiques et cerebrales) peut etre affirme par recherche moleculaire : etude familiale et detection des mutations causales, delicate en raison de l'existence de nombreux variants. Le traitement, efficace s'il est observe pendant toute la vie, doit etre biologiquement suivi (NFS, cuprurie, creatinine, proteinurie, tests hepatiques).

BACKGROUND: Premature ovarian failure (POF) is a heterogeneous syndrome, possibly due to mutations of genes involved in the normal development of the ovary and/or follicles. Based essentially on animal models, these mutations are associated with various ovarian phenotypes, from a complete absence of follicles to a partial follicular maturation. The aim of the present study was to determine whether ovarian histology, compared to pelvic ultrasonography, would be helpful in identifying which patients display an impaired follicular reserve and/or growth, and in orientating the search for POF aetiology. METHODS AND RESULTS: We studied a cohort of 61 patients suffering from POF with a normal karyotype. Their median age (range) at diagnosis was 26 years (15-39). The FSH plasma level was high, 67.0 IU/l (13-155). Estradiol and inhibin B plasma levels were low: 18.5pmol/l (18.5-555) and 5pg/ml (5-105) respectively. Both pelvic ultrasonography and ovarian biopsies were performed in each patient. The presence of follicles suggested at ultrasonography was confirmed at histology in 56% of the patients. Ovarian histology led to the distinction of two phenotypes: (i) small-sized ovaries, deprived of follicles; and (ii) normal-sized ovaries with partial follicular maturation. To confirm the value of ovarian biopsies, samples from 20 normal women were studied. These demonstrated that ovarian biopsy at random enables reliable assessment of follicular presence, especially when their size is

The thyrotropin receptor (TSHR) undergoes a cleavage at the cell membrane, leading to a heterodimer, comprising an alpha extracellular and a beta-transmembrane and intracellular subunits, held together by disulfide bonds. Moreover, part of the alpha-subunit of the receptor is shed from thyroid and transfected L cells. To understand the role of cleavage and shedding, we constructed deletion mutants starting, respectively, at the most N-terminal (S314), and C-terminal (L378) cleavage sites previously mapped, corresponding to free beta1 or beta2-subunits without further modification of receptor structure. Functional studies performed in COS-7 cells showed that both mutants display an increased basal activation of the cAMP pathway when compared with the wild-type receptor. By contrast, deletion of almost the entire extracellular domain of the receptor (TM409 mutant) totally impairs receptor function, thus confirming a role of the juxtamembrane extracellular region in receptor function. The beta1 mutant receptor exhibited an increased internalization when compared with the hormone-activated holoreceptor. Furthermore, no recycling was observed in the case of the beta1 mutant receptor. These observations strongly argue for a different conformation between the receptor activated by cleavage and shedding on the one hand, and the receptor activated by the ligand on the other hand. Cleavage and shedding of a receptor already activated by a transmembrane activating mutation M453T further increase its activity, showing that the extracellular domain still exerts a negative effect in the M453T holoreceptor. An increased internalization of the M453T receptor was observed when compared with the wild-type receptor, which was increased further in the corresponding truncated beta1-M453T receptor. Thus cleavage and shedding yield TSHR activation but also increase internalization of the free beta-subunits of the receptor, the latter mechanism limiting simultaneously excessive receptor signaling. The combined effects may be responsible for the limited basal constitutive activation of the cAMP pathway that is detected for the TSHR.

Hepatitis C virus (HCV) seroprevalence is highly diverse among human immunodeficiency virus-1 (HIV-1) infected patients, ranging between 10% of HIV-1 infected homo-bisexuel men, to92% in patients infected with HIV-1 who acquired HIV-1 through intravenous drug use. Thus, being HCV-free while having acquired HIV-1 via intravenous drug use is a rare situation. Claudin-1 is a protein involved in intracellular tight-junctions and has been identified as a major cellular co-receptor for HCV infection. Our objective was to determine whether Claudin-1 gene (CLDN1) mutations might be involved in natural resistance to HCV infection. We conducted a case-control study. All recruited patients acquired HIV-1 infection via intravenous drug use route before 1995. The case study patients remained free from HCV infection (negative anti-HCV antibodies and HCV-RNA). The control study patients was co-infected with HCV (positive anti-HCV antibodies). Direct genomic sequencing of the CLDN1 gene coding region and adjacent intron/exons junctions was performed from peripheral blood mononuclear cells. A total of 138 Caucasian patients were enrolled. Twenty-two patients (cases) were free from HCV infection and 116 (controls) were co-infected with HCV. We found single nucleotide polymorphisms (SNPs) described previously with no significant differences in allele frequencies between cases and controls. In conclusion, despite being a major cellular co-receptor for HCV entry in vitro, we did not identify any specific substitution in CLDN1 gene coding region in our study patients highly exposed but resistant to HCV infection in vivo. Other cellular co-factors involved in HCV infection should be investigated in this highly-exposed intravenous drug users patients.

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.

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.

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 α-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 devel...

Les recepteurs des gonadotrophines et de la TSH appartiennent a un sous-groupe de recepteurs couples aux proteines G qui presentent des particularites dans leur trafic cellulaire : les recepteurs de la TSH et de la FSH ont une distribution cellulaire polarisee basolaterale au niveau des cellules thyroidiennes et des cellules de Sertoli respectivement. Par contre, le recepteur de la LH est exprime de facon circonferentielle au niveau des cellules cibles gonadiques. Pour comprendre cette difference de propriete nous avons exprime ces recepteurs dans un modele de cellules epitheliales polarisees : les cellules MDCK.Nous avons ainsi montre que les trois recepteurs adoptent dans ces cellules une localisation basolaterale. Ils contiennent donc tous un signal de localisation basolaterale. Les recepteurs des gonadotrophines subissent de plus une transcytose partielle qui n’est pas observee pour le recepteur de la TSH. Nous avons montre que les proteines G heterotrimeriques jouent un role dans ce mecanisme de transcytose. Cet effet ne necessite pas une activation de l’adenylate cyclase et implique une population intracellulaire de proteines G differentes de celle qui intervient dans la transduction du signal.Nous avons ensuite identifie la nature du signal d’adressage basolateral du recepteur de la FSH par mutagenese dirigee. Nous avons montre de facon inattendue qu’il est localise dans la partie distale du domaine intracellulaire qui diverge entre les trois recepteurs. Il comprend 14 acides amines et est essentiellement dependant pour son activite d’une tyrosine et d’une leucine. Nous avons montre que ce signal est independant du signal d’endocytose du recepteur. Ce signal est autonome et transferable a une autre proteine membranaire apicale, le recepteur du NGF qu’il convertit en partie en une proteine basolaterale.Il s’agit du premier signal d’adressage basolateral caracterise pour un recepteur couple aux proteines G et plus generalement pour un recepteur hormonal.

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.

The monoclonal origin of gonadotropin-secreting pituitary adenomas has been well demonstrated but only few molecular abnormalities have so far been recognized in these tumors. For many years, several authors have suggested a role for GnRH and/or GnRH receptors (GnRH-R) in the development of these pituitary adenomas. To test the hypothesis that mutant genes encoding a constitutively activated GnRH-R might be involved in the pathogenesis of these tumors, the sequence of the GnRH-R gene was analyzed in tumoral pituitary tissue obtained from ten patients (six female, four male). The pituitary gonadotropin-secreting adenoma was associated with in vivo hypersecretion of FSH, LH and/or free alpha-subunit (n = 7) or was clinically silent (normal plasma levels of gonadotropins or free alpha-subunit, n = 3). In all cases, immunocytochemical studies of the removed adenoma confirmed their gonadotroph nature by revealing positivity for FSH, LH and/or alpha-subunit. Genomic DNA was extracted from the pathological tissue obtained at neurosurgery. Eight sequencing primers were used to amplify the three exons of the GnRH-R gene from tumoral DNA. The entire coding sequence of the GnRH-R gene was sequenced in the ten adenomas. No mutation was found in any of the tumor specimens examined. In conclusion, mutations in the GnRH receptor coding sequence occur infrequently if at all in gonadotropin-secreting pituitary adenomas.

Over the past few years, knowledge of the structure of gonadotropin receptors and their mode of action has rapidly advanced. The cDNA corresponding to the luteinizeng hormone (LH) receptor (LHR) has been cloned, leading to the identification of a novel family of G-protein-coupled receptors. The follicle stimulating hormone (FSH) receptor (FSHR) was thereafter cloned by cross-hybridization with the LHR. Structure—function relationships have been studied by mutagenesis experiments in several laboratories. The cloning and chromosomal localization to chromosome 2p21 of the two human gonadotropin receptor genes has provided insights into their evolutionary relationships. The LHR and FSHR genes are very large and contain 10 and 11 exons respectively. The obtention of monoclonal antibodies against the receptors resulted in the characterization of the receptor proteins. These antibodies also allowed the study of receptor expression in target cells in physiological and pathological conditions. The internalization of the LHR has been studied by electron microscopy. A mechanism of receptor-mediated transcytosis through the endothelial cells of the testes has been described for the LHR. The polarized expression of receptors has been studied. The cloning of gonadotropin receptor genes has opened the field of genetic study of the receptors. Inactivating mutations of the LHR have been described in Leydig cell agenesis or hypoplasia. Different phenotypes, including complete pseudohermaphroditism, ambiguous genitalia and male phenotype, have been described. In the case of the FSHR, only one mutation has been reported in familial ovarian dysgenesis with primary amenorrhea. Related males have variable alterations of spermatogenesis and fertility. Constitutive mutations of the LHR have been reported in familial testotoxicosis. One similar mutation has also been described for the FSHR. Such mutations may lead to the development of a model of receptor activation.

We report the case of an infant who presented at birth with a hypoplastic phallus associated with hypospadias. Low testosterone production, normal serum levels of steroid precursors, and increased LH in response to LH-releasing hormone supported a defect in Leydig cell differentiation or function. Conventional microscopic study of the testes showed fibroblastic cells in the interstitium. However, immunocytochemical analysis using anti-LH receptor and anti-P450c17 antibodies demonstrated that about one third of these cells were Leydig cells or precursors of Leydig cells. No histological feature could distinguish the latter cells from fibroblasts. A homozygous substitution of cysteine 133 for arginine was found in the extracellular domain of the receptor. This is the first naturally occurring missense mutation found in the extracellular domain of the LH receptor. COS-7 cells transfected with the mutant receptor exhibited a marked impairment of hCG binding, whereas some cAMP production could be observed at h...

The thyrotropin (TSH) and follicle-stimulating hormone (FSH) receptors are present mainly on the basolateral cell surface in the thyroid gland and in Sertoli cells, whereas in ovarian and in testicular cells, the luteinizing hormone (LH) receptors are distributed throughout the cell surface. When expressed in Madin-Darby canine kidney (MDCK) cells, all three receptors accumulated at the basolateral cell surface showing that they carry the corresponding targeting signals. The receptors were directly delivered to the basolateral surface of the MDCK cells. A minor fraction of the gonadotropin receptors but not of TSH receptors was secondarily targeted to the apical surface through transcytosis. The mechanisms of basolateral targeting and transcytosis were analyzed using the FSH receptor as a model. Both were insensitive to brefeldin A and pertussis toxin. Gs activation by AlF4− and cholera toxin provoked a marked enhancement of FSH receptor transcytosis. The population of Gs proteins involved in this mechanism was different from that involved in signal transduction since neither FSH nor forskolin mimicked the effects of AlF4− and cholera toxin. Gs activation provoked a similar effect on LH receptor distribution in MDCK cells, whereas it did not modify the compartmentalization of the TSH receptor. Hormone-specific transcytosis was observed in MDCK cells expressing the gonadotropin (FSH and LH) receptors and was increased after cholera toxin administration.

Objective: Familial amyloid polyneuropathy (FAP) is typically a predominantly sensory and autonomic neuropathy with progressive and late motor involvement leading to death within 10 years. Recently, prognosis was transformed with liver transplantation. Methods: We report an atypical sporadic pure motor and bulbar neuropathy initially mistaken for amyotrophic lateral sclerosis (ALS) in a 50-year-old Malian man. Results: The diagnostic procedure of this clinical purely motor and bulbar neuropathy disclosed amyloid deposits on nerve biopsy which led to the identification of a new Val93Met mutation of transthyretin. This case was also remarkable by its slow progression. Conclusions: This report confirms the motor phenotype of TTR-FAP. That should be considered in the differential diagnosis of motor neuron diseases in order to start accurate therapy. Abbreviations: ALS, amyotrophic lateral sclerosis; CIDP, chronic inflammatory demyelinating polyneuropathy; FAP, familial amyloid polyneuropathy; MRC, medical research council; PMA, progressive muscular atrophy; SALS, sporadic amyotrophic lateral sclerosis; SBMA, spino bulmbar muscular atrophy; TTR, transthyretin

Familial clustering of Graves' disease indicates a genetic etiology. Searches for genetic factors additional to the known human leukocyte antigen (HLA) association have implicated the gene for the TSH receptor (TSHR). We analyzed the linkage and association among three recently described microsatellite markers within the TSHR introns in Graves' disease in large multiply affected Welsh and English families (223 members, 44 affected individuals). Linkage analysis under a dominant model strongly rejected the hypothesis that TSHR is linked to Graves' disease in these families (lod score = -4.53). More detailed analyses also failed to provide evidence for linkage; these included combined segregation and linkage analysis, correction for HLA-DR3 status, allowance for the levels of thyroid autoantibodies in unaffected pedigree members, consideration of a recessive model for the disease, and linkage disequilibrium between disease and marker alleles. We also considered the possibility of a genetic heterogeneity of Graves' disease and thus analyzed separately the different families with a similar result. Although these results cannot eliminate a minor role of the TSHR gene locus in the genetics of Graves' disease, they argue against it being a major genetic determinant in this pathology.

Until recently, neonatal hyperthyroidism has been considered to be related to the transplacental passage of thyroid-stimulating Ig present in the serum of the mother. We report here the case of a newborn who presented with severe hyperthyroidism, diffuse goiter, and important ocular signs (eyelid retraction and possibly proptosis). However, the absence of thyroid pathology in the parents and the lack of antithyroid antibodies in the mother and in the patient led us to suspect a nonimmune aetiology. Direct genomic sequencing of the last exon of the TSH receptor in the patient revealed a T-->C transversion yielding to a Met453-->Thr heterozygous substitution in the second transmembrane domain of the receptor. The mutation was absent in both parents. Eukaryotic expression analysis in COS-7 cells yielded a mutated receptor that produced constitutive activation of adenylate cyclase without enhancement of phospholipase C activity.