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Study question: Can an infertility gene panel (GP) approach uncover genetic etiology of premature ovarian insufficiency (POI) in a patient with primary amenorrhea (PA) and hypergonadotropic hypogonadism? Summary answer: Analysis of GP using next generation sequencing (NGS) identified two new rare inactivating mutations of FSHR which are very likely responsible for the POI phenotype. What is known already: Inactivating mutations of FSHR are an extremely rare cause of POI. Sixteen inactivating mutations of the receptor have been reported so far in POI patients affecting either the extracellular domain (ECD) or the transmembrane domain (TMD) of the FSHR whether in homozygous or compound heterozygous state. In patients with previously characterized FSHR mutations, the phenotype varied depending on the level of inactivation of the receptor. In most cases, patients presented with a primary amenorrhea and streak ovaries. Some mutations were associated with partial or normal breast development, secondary amenorrhea and normal sized ovaries containing preantral and even small antral follicles. Study design, size, duration: Analysis of a panel of 31 genes implicated in POI was performed in a 32 years old Belgian patient presenting an idiopathic POI with PA diagnosed at the age of 17 and normal breast development. This patient consulted our fertility clinic for assisted reproduction with oocyte donation and gave her written informed consent to be tested for a genetic etiology of POI. Participants/materials, setting, methods: We performed GP using NGS @ BRIGHTcore in a patient with POI. FSH and AMH levels were respectively 74UI/L and 1.4ug/L. Karyotype and array CGH were normal. Transvaginal pelvic ultrasound and laparoscopy showed small ovaries with no antral follicles. Transient transfection of COS7 cells was performed with a plasmid containing wild-type FSHR cDNA as well as the two novel FSHR variants identified by GP analysis. Cell surface expression of FSHR variants was tested by FACS. Main results and the role of chance: NGS identified the presence of two new compound heterozygous FSHR missense mutations: c.646 G>A (Gly216Arg) in exon 8 (ECD) and c.1313 C>T (Thr438Ile) in exon 10 (TMD) of the receptor. Both mutations were predicted to be deleterious and/or probably damaging by in silico analysis. The in vitro functional study showed that the expression of both FSHRs variants was barely detectable at the cell surface of transfected COS7 compared to wildtype FSHR. Two experiments showed similar results. Limitations, reasons for caution: Mutations segregation in patient’s family was not evaluated. As FACS experiments used antibodies that recognize an epitope located in the ECD of FSHR, it is possible that the c.646 G>A mutation i414 Abstracts of the 34th Annual Meeting of the ESHRE, Barcelona, Spain 1 to 4 July 2018 alters the epitope recognition by the antibodies without altering the cell surface expression of the receptor. Wider implications of the findings: FSHR mutations are found in less than 1% of POI patients renderingthe single gene analysis approach inefficient. We report new FSHR inactivating mutations in a patient presenting PA and hypergonadotropic hypogonadism demonstrating the interesting contribution of a specific GP using NGS to uncover genetic etiologies of idiopathic POI cases. Trial registration number: P2016/196/CCB B406201628264 P-595 Higher resolution aneuploidy screening with targeted

International audience; To evaluate the functional role of complex asparagine-linked oligosaccharides of the human thyrotropin receptor (TSHR), a Chinese hamster ovary cell line (JP09) and a K562 cell line (K562-TSHR) expressing this receptor were treated with deoxymannojirimycin (dMM), a mannosidase I inhibitor. dMM blocks the formation of complex-type structures and leads to the formation of high-mannose-type structures. Treatment of cells with dMM led to a decrease in the number of thyrotropin (TSH)-binding sites at the cell surface. Detection of the TSHR at the cell surface using a monoclonal antibody directed against the A subunit showed that this decrease was not due to a decrease in the number of TSHRs expressed at the cell surface. However the recognition of TSHR by a monoclonal antibody directed against the C peptide was greatly decreased. On immunoblotting, after deglycosylation using peptide N-glycanase F, the A subunit was visualized as a doublet (36 and 41 kDa). In control cells the species of higher molecular mass was more abundant whereas after dMM treatment the species of lower molecular mass became more abundant. This difference in molecular mass between the two peptides is compatible with the removal of the C peptide. In conclusion, the results show that inhibition of complex-type structure formation leads to (i) an incapacity for TSHR to bind TSH, without affecting its intracellular transport and (ii) an increase of TSHR susceptibility to proteases that remove the C peptide. We then hypothesized that removal of the C peptide could contribute to the formation of a non-functional TSHR.

The generation of Abs recognizing the native structure of the human thyrotropin receptor (hTSHR) has been difficult because there is currently no method allowing the purification of correctly folded Ag in the amounts required by classical immunization protocols. The majority of Abs made against the hTSHR react preferentially with denatured molecules. We report that a humoral response against the native hTSHR, compatible with mAb production, is elicited in mice by immunization with a DNA construct encoding the receptor. BALB/c mice were inoculated in the anterior tibialis muscle with 100 μg of plasmid DNA harboring the hTSHR cDNA. Eleven weeks after the first injection, 10 mice of 14 showed by FACS analysis a strong IgG response against the hTSHR expressed at the surface of Chinese hamster ovary cells. A clear TSH-binding inhibiting Ig and thyrotropin-blocking Ab activity (competition with TSH binding and TSH activity, respectively) was demonstrated in the majority of sera tested. One serum exhibited a clear stimulating activity. Despite the maintenance of normal circulating free T4 levels in all mice, these bioactivities persisted until 18 wk, in which mice were sacrificed, their thyroids were examined histologically, and spleens from two animals were used for mAb production. All mice displayed a severe lymphocytic infiltration of their thyroids, composed mostly of activated B cells. Three mAbs were produced against conformational epitopes of the hTSHR. We conclude that genetic immunization is an efficient method of generating Abs recognizing the native structure of the hTSHR and a new way of inducing thyroiditis in mice murine.

Ovarian hyperstimulation syndrome (OHSS) occurs mainly after excessive stimulation of the ovaries by exogenous gonadotropins administrated in the context of in vitro fertilization procedures (iatrogenic OHSS). Recently, spontaneous and recurrent occurrence of the disease (spontaneous OHSS) was shown in two families to be caused by mutations affecting the follitropin receptor (FSHr). The two mutant FSHr (T449I, D567N) harbor aminoacid substitutions in the serpentine portion of the receptor and display abnormally high sensitivity to the pregnancy hormone hCG, thus providing a satisfactory explanation to the phenotype. In addition, mutant D567N showed also increased sensitivity to thyrotopin (TSH) and displayed increase in basal (ligand-independent) activity. In this report, we describe a new familial case of recurrent OHSS. The affected women were heterozygous for a different mutation involving codon 449, where an alanine was substituted for threonine. Similar to D567N, the T449A FSHr mutant shows an increase of its sensitivity to both hCG and TSH, together with an increase in basal activity. Together with the two previous studies, this report shows that inappropriate stimulation of the FSHr by hCG is a cause of spontaneous OHSS.

Germline loss-of-function mutations of TSH receptor (TSHR) gene have been described in families with partial or complete TSH resistance. Large TSH elevations were generally found in the patients with homozygous or compound heterozygous mutations. In this study, we sequenced the entire TSHR gene in a series of 10 unrelated patients with slight (6.6-14.9 mU/liter) to moderate (24-46 mU/liter) elevations of serum TSH, associated with definitely normal free thyroid hormone concentrations. Thyroid volume was normal in all patients, except two with a modest hypoplasia. Autoimmune thyroid disease was excluded in all patients on the basis of clinical and biochemical parameters. Eight patients had at least one first-degree relative bearing the same biochemical picture. TSHR mutations were detected in 4 of 10 cases by analyzing DNA from peripheral leukocytes. A compound heterozygosity (P162A on maternal allele, and the novel mutation C600R on the paternal one) was found in the patient with the highest TSH levels. Only one TSHR allele was mutated in the remaining three cases, and no alterations in TSHR gene promoter were detected in all of these probands. A novel mutation (L467P) was detected on the maternal allele in one patient and in her monozygotic twin. Previously described inactive mutants, T655Delta and C41S, were detected in the other two cases. When tested on several occasions, circulating TSH values fluctuating above the upper limit of the normal range could be shown in heterozygous subjects of these families. A dominant mode of inheritance of the biochemical alterations was detected in these cases. Mutant TSHRs were studied during transient expression in COS7 and HEK293T cells. Their TSH-independent cAMP accumulation activities were very low or similar to mock-transfected cells, and no increases were seen after maximal hormone stimulation. Flow cytometry experiments showed a poor level of expression of all mutant TSHRs at the cell membrane. In conclusion, we found several loss-of-function mutations of TSHR, including two novel ones, in a series of unrelated patients with slightly elevated TSH levels. Therefore, partial resistance to TSH action is a frequent finding among patients with slight hyperthyrotropinemia of nonautoimmune origin. Germline mutations of TSHR may be associated with serum TSH values fluctuating above the upper limit of the normal range, also in the heterozygous state

Congenital hypothyroidism (CH) is a relatively frequent and potentially severe disease. It is classically subdivided into: 1) thyroid dysgenesis (TD), a defect in the organogenesis of the gland leading to hypoplastic, ectopic, or absent thyroid gland; or 2) thyroid dyshormonogenesis, a defect in one of the biochemical mechanisms responsible for thyroid hormone synthesis. Most cases of TD are sporadic, although familial occurrences have occasionally been described. Recently, several genes have been implicated in a small proportion of TD, but, in the majority of the cases, the etiology remains unknown. PAX8 is a transcription factor involved in thyroid development. So far, three loss-of-function mutations of PAX8 have been described, two in sporadic cases and one in familial thyroid hypoplasia. Here, we describe a novel mutation of PAX8 causing autosomal dominant transmission of CH with thyroid hypoplasia. The mutation consists of the substitution of a tyrosine for cysteine 57 in the paired domain of PAX8. When tested in cotransfection experiments with a thyroid peroxidasse promoter construct, the mutant allele was unable to exert its normal transactivation effect on transcription. Our results give further evidence that, contrary to the situation in knockout mice, haplo-insufficiency of PAX8 is a cause of CH in humans.

Metastatic thyroid carcinoma rarely provokes hyperthyroidism. We describe here the main characteristics of this association observed in a 48-year-old woman.The patient had presented signs of hyperthyroidism for one year. Clinical examination revealed severe Graves' disease with nodular goiter. Presurgery investigations demonstrated thyroid carcinoma and lung metastasis. Thyroid stimulating immunoglobulins were elevated. After total thyroidectomy, hyperthyroidism persisted, indicating the functional nature of the lung metastases.The effect of thyroid stimulating immunoglobulins on the clinical course of thyroid carcinoma and distant metastases remains unknown.

In this paper we report the isolation of a self-reactive cytotoxic gamma delta T cell line, 158RE.2, that originates from the T lymphocyte population infiltrating the thyroid gland of a patient with Graves' disease. Functional data using this cell line demonstrate that gamma delta T cells expanded in the thyroid tissue specifically recognize a ligand expressed by thyroid epithelial cells and cell lines of endocrine epithelial origin. The TCR expressed by these gamma delta T cells--V gamma I/V delta 5--is unusual in peripheral blood lymphocytes, and its specificity is clearly different from that observed in a high percentage of gamma delta T cells from PBL, which express the common TCR V gamma 9/V delta 2. The V gamma I/V delta 5 receptor is involved in the recognition of the ligand expressed by the thyroid cells, but not in the NK-like activity also displayed by 158RE.2. These cells express CD8 alpha alpha dimers, which participate in the thyroid ligand recognition but not in the NK-like activity. The epithelial cell recognition is not restricted by classical MHC class I or class II molecules, although the CD8 alpha alpha participation in the recognition suggests the involvement of nonclassical MHC molecules. These are the first data to be presented on self-reacting gamma delta T cells in human epithelium.