Female transgenic mice that overexpress the human chorionic gonadotrophin β subunit (hCGβ+) develop prolactinomas, whereas hCGβ+ males do not. The high levels of circulating hCG induce massive ...luteinization in the ovary of hCGβ+ females, and progesterone becomes the primary steroid hormone produced, but estradiol remains at physiological level. The involvement of high levels of progesterone in lactotroph proliferation is not clearly understood; hence, the pathogenesis of prolactinomas in hCGβ+ females remains unclear. TGFβ1 is an inhibitor of lactotroph function, and the reduced TGFβ1 activity found in prolactinomas has been proposed to be involved in tumor development. The aim of the present work was to study the role of TGFβ1 in the gender-specific development of prolactinomas in hCGβ+ mice. We compared the expression of different components of the pituitary TGFβ1 system in males and females in this model. We found reduced TGFβ1 levels, reduced expression of TGFβ1 target genes, TGFβ1 receptors, Ltbp1, Smad4 and Smad7 in hCGβ+ female pituitaries. However, no differences were found between the transgenic and wild-type male pituitaries. We postulate that decreased pituitary TGFβ1 activity in hCGβ+ females is involved in the development of prolactinomas. In fact, we demonstrated that an in vivo treatment carried out for increasing pituitary TGFβ1 activity, was successful in reducing the prolactinoma development, and the hyperprolactinemia in hCGβ+ females. Moreover, the stronger TGFβ1 system found in males could protect them from excessive lactotroph proliferation. Sex differences in the regulation of the pituitary TGFβ1 system could explain gender differences in the incidence of prolactinoma.
Abstract
Hypopituitarism with deficiency of one or more pituitary hormones (combined pituitary hormone deficiency or CPHD) can vary in severity and age at presentation. Additionally, the hormone ...abnormalities may evolve with time necessitating frequent evaluation. These hormonal deficits can also be present as part of a syndrome, with patients showing extrapituitary abnormalities such as eye or forebrain malformations. Over the past decade, there has been an explosion in the knowledge of the genetic cascade that orchestrates hypothalamic and pituitary development. Several transcription factors and signaling molecules are critical for cell differentiation and proliferation at a very early stage of gestation. However, more genes remain to be identified in order to provide patients with a definitive aetiology. We studied 95 children with congenital hypopituitarism from seven pediatric hospitals from Argentina. Children with non-endocrine, non-familial idiopathic short stature served as a control group (n=100). A custom exon capture panel based on single-molecule molecular inversion probes sequencing (MIPS) was performed in probands and parents. This panel tests 104 genes: including a selection of reported and candidate genes chosen from our knowledge of pituitary development in mice and PROP1 interacting proteins. We found at least 1 variant in 50 probands. The prevalence of known variants in the pituitary transcription factor genes PROP1 and PIT1 (frequently mutated in CPHD) was low in our cohort. A significant number of disease-causing variants were found in LHX3, LHX4, GLI2 and OTX2. An important advance of our study is the identification of pathogenic variants in recently discovered and novel genes. We found an heterozygous variant in the Roundabout (ROBO) receptor homolog 1 gene: ROBO1;p.Arg1504* in a patient with CPHD and septo-optic dysplasia (SOD); two variants in Steroid Receptor SRA1: p. Ile179Thr and p.Val110delinsArgLeu in a patient with CPHD and hypogonadism hypogonadotropic and a missense variant in Semaphorin 3A SEMA3A: p.Val588Leu in a case of CPHD and aortic coarctation. Our findings support evidence that the etiology of congenital hypopituitarism is highly heterogeneous and may be infrequently monogenic with full penetrance, underlying a more complex pathogenesis.
Presentation: Sunday, June 12, 2022 12:36 p.m. - 12:41 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.
Serum prolactin levels gradually increase from birth to puberty in both male and female rats, with higher levels observed in female since the first days of life. The increase in lactotroph secretion ...was attributed to the maturation of prolactin-inhibiting and prolactin-releasing factors; however, those mechanisms could not fully explain the gender differences observed. Prolactin secretion from isolated lactotrophs, in the absence of hypothalamic control, also increases during the first weeks of life, suggesting the involvement of intra-pituitary factors. We postulate that pituitary transforming growth factor beta 1 (TGFβ1) is involved in the regulation of prolactin secretion as well as in the gender differences observed at early postnatal age. Several components of the local TGFβ1 system were evaluated during postnatal development (11, 23, and 45 days) in female and male Sprague–Dawley rats. In vivo assays were performed to study local TGFβ1 activation and its impact on prolactin secretion. At day 11, female pituitaries present high levels of active TGFβ1, concomitant with the highest expression of TGFβ1 target genes and the phospho-Smad3 immunostaining in lactotrophs. The steady increase in prolactin secretion inversely correlates with active TGFβ1 levels only in females. Dopamine and estradiol induce TGFβ1 activation at day 11, in both genders, but its activation induces the inhibition of prolactin secretion only in females. Our findings demonstrate that: (1) TGFβ1 activation is regulated by dopamine and estradiol; (2) the inhibitory regulation of local TGFβ1 on prolactin secretion is gender specific; and (3) this mechanism is responsible, at least partially, for the gender differences observed being relevant during postnatal development.
Abstract
Pituitary hormone deficiency or hypopituitarism is characterized by a malformed or underdeveloped pituitary gland resulting in an impaired pituitary hormone secretion. Several transcription ...factors have been described in its etiology, but defects in known genes account for only a small proportion of cases. We sought to identify the cause of hypopituitarism in 171 unrelated patients diagnosed with or without extra-pituitary manifestations that were recruited from several Argentinean medical centers. We conducted panel sequencing, and identified among other genes and variants, de novo heterozygous mutations in FOXA2 and PNPLA6. FOXA2 is a transcription factor member of the forkhead class of DNA-binding proteins, involved in the early development of multiple tissues. FOXA2 is highly expressed throughout the developing hypothalamic-pituitary axis, and regulates GLI2, SHH and NKX2-2 expression. Mutations of FOXA2 have been linked to combined pituitary hormone deficiency (CPHD) in some cases with extra-pituitary phenotypes including hyperinsulinism or gastrointestinal malformations. We found two patients with CPHD and rare FOXA2 variants. Case 1 had GHD, anterior pituitary hypoplasia, mammary hypertelorism and digital anomalies and a heterozygous variant FOXA2 p.Arg228Ser, predicted to be pathogenic. Case 2 had GH and TSH deficiency, craniofacial anomalies and neurodevelopmental delay, and a novel, stop codon mutation FOXA2 p.Ser229* and an heterozygous GLI1 variant (p.Asp1048Asn). Both FOXA2 variants are located within the forkhead domain which may affect the DNA binding ability. We suspect they are likely damaging based on the literature, the in-silico prediction, and their absence in GnomAD. PNPLA6 is a conserved lysophospholipase involved in maintaining nervous system integrity. Mutations in PNPLA6 have been identified in a broad spectrum from pure ataxia to rare neuroendocrine conditions including Gordon Holmes and Oliver McFarlane syndromes. Here, we identified two de novo heterozygous variants in PNPLA6 in children with CPHD. Variant p.W1039R was found in a patient with CPHD, intellectual disability and visual problems. A second variant (p.T1115P) was identified in a 10-year-old girl with CPHD, retinitis pigmentosa and neurodevelopmental delay. According to modelling studies of the protein structure, both variants are expected to be critical for the activity of the NTE as they are located in close proximity to the protein’s catalytic pocket. It is likely that these variants may contribute to our patient’s phenotype. However, as most reported PNPLA6 variants in the literature were found in homozygosity or compound heterozygosity, additional studies are necessary to draw more definitive genotype-phenotype correlations. In summary, in this work we were able to expand our knowledge of pituitary target genes for genetic diagnosis for CH.
Abstract
Congenital hypopituitarism (CH), septo-optic dysplasia (SOD), and holoprosencephaly (HPE) constitute an important group of structural birth defects that cause significant morbidity and ...life-long consequences for quality of life and care. The genetic causes are highly overlapping. As such, these disorders can be considered as a spectrum of related disorders. Improved insight into genetic causes would be valuable for patients, families, and medical geneticists. Very few systematic genetic screens have been carried out for patients with CH. We implemented genetic screening using single-molecule molecular inversion probes sequencing to identify causative mutations in a set of 67 genes previously reported in CH patients and the spectrum encompassing SOD and HPE. We captured genomic DNA from 170 Argentinean pediatric patients with CH, and 54% of the patients in this cohort have craniofacial, ophthalmologic, and/or central nervous system defects. We found candidate pathogenic, likely pathogenic and variants uncertain significance (VUS) in 23% of the cases. In order to evaluate the functional consequences of VUS in LHX3, LHX4, and GLI2, we performed in-vitro functional assays to study the activity of the mutated proteins. To test LHX3/4 variants we co-transfected HEK293T cells with wild type (WT) or mutated LHX3/4 variant plasmids and luciferase reporter genes driven by the ɑGSU promoter or GH1 promoter and assayed for luciferase activity. For GLI2 functional analysis we used the cell line NIH/3T3-CG, stably transfected to express GFP under the presence of GLI2 activated form. Endogenous Gli2 was knocked out by CRISPR-Cas9 and clones were selected for absence of GFP expression upon activation of the sonic hedgehog pathway. We tested the ability of transfected WT or mutated GLI2 expression plasmids to restore GFP fluorescence. We concluded that variants LHX3:p.Pro187Ser LHX4:p.Arg84His, p.Gln100His and p.Trp204Leu and GLI2:p.1404Lfs impair activation of the reporter gene, while the LHX3:p.Leu220Met and GLI2:p.L761P have WT activity on their respective assays. Identification of disease-causing variants in CH is complicated by phenotypic variation, incomplete penetrance, and VUS. Functional testing of potentially pathogenic variants is critical to arrive at a definitive molecular diagnosis. A full catalogue of variant effects in known causative genes would be invaluable for clinicians in order to simplify the interpretation of novel variants and reduce the diagnostic odyssey that families often experience.
The unreinforced masonry (URM) buildings designed to be conforming with the Italian building code, as illustrated in the companion paper, were analyzed by performing time-history analyses on models ...realized using an equivalent frame approach and by adopting two different constitutive laws. Both the effect of record-to-record variability and of epistemic and aleatory uncertainties in modelling were explored. The achieved results constitute the basis for the evaluation of the risk level implicit in Italian code-conforming buildings. Two main performance conditions are considered, namely usability-preventing damage and global collapse limit states.
New Italian data on the distribution of the Annex I Habitats are reported in this contribution. Specifically, 8 new occurrences in Natura 2000 sites are presented and 49 new cells are added in the ...EEA 10 km × 10 km reference grid. The new data refer to the Italian administrative regions of Campania, Calabria, Marche, Piedmont, Sardinia, Sicily, Tuscany and Umbria. Relevés and figures are provided as Supplementary material respectively 1 and 2.
Neoadjuvant chemotherapy (NAC) has a profound impact on surgical management of breast cancer. For this reason, the Italian Association of Breast Surgeons (ANISC) promoted the third national Consensus ...Conference on this subject, open to multidisciplinary specialists.
The Consensus Conference was held on-line in November 2022, and after an introductory session with five core-team experts, participants were asked to vote on eleven controversial issues, while results were collected in real-time with a polling system.
A total of 164 dedicated specialists from 74 Breast Centers participated. Consensus was reached for only three of the eleven issues, including: 1) the indication to assess the response with Magnetic Resonance Imaging (79 %); 2) the need to re-assess the biological factors of the residual tumor if present (96 %); 3) the possibility of omitting a formal axillary node dissection for cN1 patients if a pathologic Complete Response (pCR) was confirmed with analysis of one or more sentinel lymph nodes (82 %). The majority voted in favor of mapping both the breast and nodal lesions pre-NAC (59 %), and against the omission of sentinel lymph node biopsy in cN0 patients in the case of pathologic or clinical Complete Response (69 %). In cases of cT3/cN1+ tumors with pCR, only 8 % of participants considered appropriate the omission of Post-Mastectomy Radiation Therapy.
There is still a wide variability in surgical approaches after NAC in the “real world”. As NAC is increasingly used, multidisciplinary teams should be attuned to conforming their procedures to the rapid advances in this field.
CHD is one of the most common serious chronic conditions in postmenopausal women and leads to extremely high risk for recurrent myocardial infarction and death. On the basis of the currently ...available randomized clinical-trial results the role of conventional HRT for treatment and prevention of CHD is rapidly evolving from presumed benefit to proven harm, at least in some categories of women yet to define. For this reason there has been a particular interest in potential clinical uses of selective estrogen receptor modulators (SERMs). SERMs are a class of compounds that can act as estrogen receptor (ER) agonists in some domains (bone and lipids) and acting as ER antagonists in others (breast and uterus). Raloxifene hydrochloride is an antiestrogen that is currently approved only to treat osteoporosis in postmenopausal women. Because of its effects on lipids and other biomarkers of cardiovascular risk, there is great interest in determining whether it may benefit the cardiovascular system. The great majority of data on cardiovascular effects of raloxifene concern effects on lipids and markers of thrombosis and inflammation. The purpose of this review is to summarize the best available evidence concerning raloxifene and cardiovascular disease focusing some areas known to be important risk factors for cardiovascular diseases: lipids and lipoproteins, glucose metabolism, hemostatic factors, markers of inflammation and cardiovascular function.
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