Contrasting Phenotypes in Resistance to Thyroid Hormone Alpha Correlate with Divergent Properties of Thyroid Hormone Receptor α1 Mutant Proteins.

BACKGROUND: Resistance to thyroid hormone alpha (RTHα), a disorder characterized by tissue-selective hypothyroidism and near-normal thyroid function tests due to thyroid receptor alpha gene mutations, is rare but probably under-recognized. This study sought to correlate the clinical characteristics and response to thyroxine (T4) therapy in two adolescent RTHα patients with the properties of the THRA mutation, affecting both TRα1 and TRα2 proteins, they harbored. METHODS: Clinical, auxological, biochemical, and physiological parameters were assessed in each patient at baseline and after T4 therapy. RESULTS: Heterozygous THRA mutations occurring de novo were identified in a 17-year-old male (patient P1; c.788C>T, p.A263V mutation) investigated for mild pubertal delay and in a 15-year-old male (patient P2; c.821T>C, p.L274P mutation) with short stature (0.4th centile), skeletal dysplasia, dysmorphic facies, and global developmental delay. Both individuals exhibited macrocephaly, delayed dentition, and constipation, together with a subnormal T4/triiodothyronine (T3) ratio, low reverse T3 levels, and mild anemia. When studied in vitro, A263V mutant TRα1 was transcriptionally impaired and inhibited the function of its wild-type counterpart at low (0.01-10 nM) T3 levels, with higher T3 concentrations (100 nM-1 μM) reversing dysfunction and such dominant negative inhibition. In contrast, L274P mutant TRα1 was transcriptionally inert, exerting significant dominant negative activity, only overcome with 10 μM of T3. Mirroring this, normal expression of KLF9, a TH-responsive target gene, was achieved in A263V mutation-containing peripheral blood mononuclear cells following 1 μM of T3 exposure, but with markedly reduced expression levels in L274P mutation-containing peripheral blood mononuclear cells, even with 10 μM of T3. Following T4 therapy, growth, body composition, dyspraxia, and constipation improved in P1, whereas growth retardation and constipation in P2 were unchanged. Neither A263V nor L274P mutations exhibited gain or loss of function in the TRα2 background, and no additional phenotype attributable to this was discerned. CONCLUSIONS: This study correlates a milder clinical phenotype and favorable response to T4 therapy in a RTHα patient (P1) with heterozygosity for mutant TRα1 exhibiting partial, T3-reversible, loss of function. In contrast, a more severe clinical phenotype refractory to hormone therapy was evident in another case (P2) associated with severe, virtually irreversible, dysfunction of mutant TRα1

Internações por aborto no Brasil, 2008-2018: estudo ecológico de série temporal

Objective: To analyze the temporal trend of hospitalizations caused by abortion in Brazil between 2008-2018, according to Region and federal units (FU). Methods: Ecological study concerning hospitalizations due to abortion of women of childbearing age, from Hospital Information System data. The rates were calculated according the characteristics of women. The trend was assessed by Prais-Winsten generalized linear regression. Results: The 2,258,104 hospitalizations due to abortion 4 represent 5% of all hospitalizations in women of childbearing age. Hospitalizations for abortions in Brazil reduced 0.76 in the period; this trend occurred in 19 Brazilian FUs and all regions, except the South (stable). There was a significant reduction (p<0.001) in hospitalizations for spontaneous abortion and in admissions for abortion in women aged 20 to 39 years. Conclusion: A temporal trend of reduction in the hospitalizations due to abortion in Brazil was found, variating according to characteristics of the woman, FU and Region of residence.Objetivo: Analizar la tendencia temporal de las hospitalizaciones por aborto en Brasil de 2008-2018, según región y unidad federativa. Métodos: Estudio ecológico con datos sobre hospitalizaciones por aborto de mujeres en edad fértil del Sistema de Información Hospitalaria. Las tasas se calcularon según las características de la mujer y la tendencia se evaluó mediante regresión lineal generalizada de Prais-Winsten. Resultados: Las 2.258.104 hospitalizaciones por aborto representan el 5% de todas las hospitalizaciones en mujeres en edad fértil. Hubo una reducción significativa de 0,76 puntos porcentuales por año; esta tendencia ocurrió en 19 UF brasileñas y en todas las regiones, excepto en el Sur (estable). Hubo una reducción significativa (p<0,001) en las hospitalizaciones por aborto espontáneo y en mujeres de 20 a 39 años. Conclusión: El estudio mostró una tendencia a reducir las hospitalizaciones por aborto en Brasil, con variaciones según características de la mujer, UF y Región de residenciaObjetivo: Analisar a tendência temporal das internações por aborto no Brasil, de 2008 a 2018, segundo região e Unidades da Federação (UF). Métodos: Estudo ecológico, com dados de internações por aborto de mulheres em idade fértil registrados no Sistema de Informações Hospitalares/Sistema Único de Saúde (SIH/SUS). As taxas foram calculadas segundo características da mulher; e a tendência, avaliada por regressão linear generalizada de Prais-Winsten.  Resultados: As 2.258.104 internações por aborto representaram 5% de todas as internações de mulheres em idade fértil. Houve redução significativa, de 0,76 pontos percentuais ao ano, no período. Essa tendência ocorreu em 19 UFs brasileiras e em todas as regiões, exceto a Sul (estável). Houve redução significativa (p-valor<0,001) nas internações por aborto espontâneo e nas internações de mulheres de 20 a 39 anos. Conclusão: Observou-se tendência de redução das internações por aborto no país, com variações segundo características da mulher, UF e região de residência

A novel albumin gene mutation (R222I) in familial dysalbuminemic hyperthyroxinemia.

CONTEXT: Familial dysalbuminemic hyperthyroxinemia, characterized by abnormal circulating albumin with increased T4 affinity, causes artefactual elevation of free T4 concentrations in euthyroid individuals. OBJECTIVE: Four unrelated index cases with discordant thyroid function tests in different assay platforms were investigated. DESIGN AND RESULTS: Laboratory biochemical assessment, radiolabeled T4 binding studies, and ALB sequencing were undertaken. (125)I-T4 binding to both serum and albumin in affected individuals was markedly increased, comparable with known familial dysalbuminemic hyperthyroxinemia cases. Sequencing showed heterozygosity for a novel ALB mutation (arginine to isoleucine at codon 222, R222I) in all four cases and segregation of the genetic defect with abnormal biochemical phenotype in one family. Molecular modeling indicates that arginine 222 is located within a high-affinity T4 binding site in albumin, with substitution by isoleucine, which has a smaller side chain predicted to reduce steric hindrance, thereby facilitating T4 and rT3 binding. When tested in current immunoassays, serum free T4 values from R222I heterozygotes were more measurably abnormal in one-step vs two-step assay architectures. Total rT3 measurements were also abnormally elevated. CONCLUSIONS: A novel mutation (R222I) in the ALB gene mediates dominantly inherited dysalbuminemic hyperthyroxinemia. Susceptibility of current free T4 immunoassays to interference by this mutant albumin suggests likely future identification of individuals with this variant binding protein.This work was supported by funding from the Wellcome Trust (Grant 100585/Z/12/Z, to N.S., Grant 095564/Z/11/Z, to K.C.) and National Institute for Health Research Cambridge Biomedical Research Centre (to C.M., and M.G.).This is the final published version of the article. It was originally published in The Journal of Clinical Endocrinology & Metabolism (Nadia Schoenmakers, Carla Moran, Irene Campi, Maura Agostini, Olivia Bacon, Odelia Rajanayagam, John Schwabe, Sonia Bradbury, Timothy Barrett, Frank Geoghegan, Maralyn Druce, Paolo Beck-Peccoz, Angela O'Toole, Penelope Clark, Michelle Bignell, Greta Lyons, David Halsall, Mark Gurnell, Krishna Chatterjee. J Clin Endocrinol Metab 2014 Jul 19;99(7):E1381-6. Epub 2014 Mar 19. http://dx.doi.org/10.1210/jc.2013-4077). A correction to this article was issued because the CC-BY licence was not present on the final published paper (http://dx.doi.org/10.1210/jc.2015-1656)

A Pharmacogenetic Approach to the Treatment of Patients With PPARG Mutations.

Loss-of-function mutations in PPARG cause familial partial lipodystrophy type 3 (FPLD3) and severe metabolic disease in many patients. Missense mutations in PPARG are present in ∼1 in 500 people. Although mutations are often binarily classified as benign or deleterious, prospective functional classification of all missense PPARG variants suggests that their impact is graded. Furthermore, in testing novel mutations with both prototypic endogenous (e.g., prostaglandin J2 [PGJ2]) and synthetic ligands (thiazolidinediones, tyrosine agonists), we observed that synthetic agonists selectively rescue function of some peroxisome proliferator-activated receptor-γ (PPARγ) mutants. We report on patients with FPLD3 who harbor two such PPARγ mutations (R308P and A261E). Both PPARγ mutants exhibit negligible constitutive or PGJ2-induced transcriptional activity but respond readily to synthetic agonists in vitro, with structural modeling providing a basis for such differential ligand-dependent responsiveness. Concordant with this finding, dramatic clinical improvement was seen after pioglitazone treatment of a patient with R308P mutant PPARγ. A patient with A261E mutant PPARγ also responded beneficially to rosiglitazone, although cardiomyopathy precluded prolonged thiazolidinedione use. These observations indicate that detailed structural and functional classification can be used to inform therapeutic decisions in patients with PPARG mutations

Anemia in Patients With Resistance to Thyroid Hormone α: A Role for Thyroid Hormone Receptor α in Human Erythropoiesis

Context: Patients with resistance to thyroid hormone (TH) α (RTHα) are characterized by growth retardation, macrocephaly, constipation, and abnormal thyroid function tests. In addition, almost all RTHα patients have mild anemia, the pathogenesis of which is unknown. Animal studies suggest an important role for TH and TH receptor (TR)α in erythropoiesis.Objective: To investigate whether a defect in TRα affects the maturation of red blood cells in RTHα patients.Design, Setting, and Patients: Cultures of primary human erythroid progenitor cells (HEPs), from peripheral blood of RTHα patients (n = 11) harboring different inactivating mutations in TRα (P398R, F397fs406X, C392X, R384H, A382fs388X, A263V, A263S), were compared with healthy controls (n = 11). During differentiation, erythroid cells become smaller, accumulate hemoglobin, and express different cell surface markers. We assessed cell number and cell size, and used cell staining and fluorescence-activated cell sorter analysis to monitor maturation at different time points.Results: After ∼14 days of ex vivo expansion, both control and patient-derived progenitors differentiated spontaneously. However, RTHα-derived cells differentiated more slowly. During spontaneous differentiation, RTHα-derived HEPs were larger, more positive for c-Kit (a proliferation marker), and less positive for glycophorin A (a differentiation marker). The degree of abnormal spontaneous maturation of RTHα-derived progenitors did not correlate with severity of underlying TRα defect. Both control and RTHα-derived progenitors responded similarly when differentiation was induced. T3 exposure accelerated differentiation of both control- and RTHα patient-derived HEPs.Conclusions: Inactivating mutations in human TRα affect the balance between proliferation and differentiation of progenitor cells d

Prospective functional classification of all possible missense variants in PPARG.

Clinical exome sequencing routinely identifies missense variants in disease-related genes, but functional characterization is rarely undertaken, leading to diagnostic uncertainty. For example, mutations in PPARG cause Mendelian lipodystrophy and increase risk of type 2 diabetes (T2D). Although approximately 1 in 500 people harbor missense variants in PPARG, most are of unknown consequence. To prospectively characterize PPARγ variants, we used highly parallel oligonucleotide synthesis to construct a library encoding all 9,595 possible single-amino acid substitutions. We developed a pooled functional assay in human macrophages, experimentally evaluated all protein variants, and used the experimental data to train a variant classifier by supervised machine learning. When applied to 55 new missense variants identified in population-based and clinical sequencing, the classifier annotated 6 variants as pathogenic; these were subsequently validated by single-variant assays. Saturation mutagenesis and prospective experimental characterization can support immediate diagnostic interpretation of newly discovered missense variants in disease-related genes.This work was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (1K08DK102877-01, to A.R.M.; 1R01DK097768-01, to D.A.), NIH/Harvard Catalyst (1KL2TR001100-01, to A.R.M.), the Broad Institute (SPARC award, to A.R.M. and T.M.), and the Wellcome Trust (095564, to K.C.; 107064, to D.B.S.).This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/ng.370

Anemia in Patients With Resistance to Thyroid Hormone α: A Role for Thyroid Hormone Receptor α in Human Erythropoiesis

Context: Patients with resistance to thyroid hormone (TH) α (RTHα) are characterized by growth retardation, macrocephaly, constipation, and abnormal thyroid function tests. In addition, almost all RTHα patients have mild anemia, the pathogenesis of which is unknown. Animal studies suggest an important role for TH and TH receptor (TR)α in erythropoiesis. Objective: To investigate whether a defect in TRα affects the maturation of red blood cells in RTHα patients. Design, Setting, and Patients: Cultures of primary human erythroid progenitor cells (HEPs), from peripheral blood of RTHα patients (n = 11) harboring different inactivating mutations in TRα (P398R, F397fs406X, C392X, R384H, A382fs388X, A263V, A263S), were compared with healthy controls (n = 11). During differentiation, erythroid cells become smaller, accumulate hemoglobin, and express different cell surface markers. We assessed cell number and cell size, and used cell staining and fluorescence-activated cell sorter analysis to monitor maturation at different time points. Results: After ∼14 days of ex vivo expansion, both control and patient-derived progenitors differentiated spontaneously. However, RTHα-derived cells differentiated more slowly. During spontaneous differentiation, RTHα-derived HEPs were larger, more positive for c-Kit (a proliferation marker), and less positive for glycophorin A (a differentiation marker). The degree of abnormal spontaneous maturation of RTHα-derived progenitors did not correlate with severity of underlying TRα defect. Both control and RTHα-derived progenitors responded similarly when differentiation was induced. T3 exposure accelerated differentiation of both control- and RTHα patient-derived HEPs. Conclusions: Inactivating mutations in human TRα affect the balance between proliferation and differentiation of progenitor cells during erythropoiesis, which may contribute to the mild anemia seen in most RTHα patients.A.L.M.v.G., M.E.M., and R.P.P. are supported by ZonMWTOP Grant 91212044 and an Erasmus MC Medical Research Advisory Committee (MRACE) grant. A.L.M.v.G. and R.P.P. are also supported by a European Thyroid Association (ETA) research grant. K. Chatterjee is supported by Wellcome Trust Investigator Award 095564/Z/11/Z. K. Chatterjee and C.M. are supported by the National Institute for Health Research Cambridge Biomedical Research Centre

Molecular Analysis of Thyroid Hormone Receptor Beta and Peroxisome Proliferator-Activated Receptor Gamma Action

The nuclear receptor superfamily comprises a group of ligand-activated transcription factors that regulate the expression of target genes. They play a central role in diverse physiological pathways, and are therefore extremely important in the aetiology of various human disorders and as pharmaceutical therapeutic targets. This thesis describes molecular analyses of the thyroid hormone receptor (TR) and the peroxisome proliferator-activated receptor gamma (PPARγ), in disorders of thyroid hormone and insulin action respectively. The syndrome of Resistance to Thyroid Hormone (RTH), characterized by reduced tissue responsiveness to circulating thyroid hormones, is associated with diverse mutations in the ligand-binding domain of the thyroid hormone β receptor, localizing to three clusters around the hormone binding cavity. The first part of this thesis describes three novel RTH mutations (S314C, S314F, S314Y), due to different amino acid substitutions in the same codon, occurring in six separate families. Characterization of these mutant receptors showed marked differences in their functional impairment. In the second part of the thesis I report detailed functional studies of natural and synthetic receptor agonists with loss-of-function PPARγ mutants (P467L; V290M), previously identified in patients with severe insulin resistance, type 2 diabetes mellitus and hypertension. Both PPARγ mutants act as dominant negative inhibitors of wild type receptor (WT) action because of their failure to fully dissociate from corepressors. My results provide evidence that tyrosine-based rather than thiazolidinedione PPARγ agonists, may represent a more rational therapeutic approach to restoring mutant receptor function and ameliorating insulin resistance in our patients. Then, in an unrelated kindred a different, digenic mechanism of insulin resistance, with a combination of loss-of-function mutations in PPARγ and PPP1R3 (muscle-specific subunit of protein-phosphatase 1 mediating glycogen synthesis) is described. Functional characterisation of these mutant proteins provides unique insights into the complex interplay between this nuclear receptor and a second metabolic signalling pathway. Finally, three novel heterozygous mutations, in the ligand and DNA binding domains of PPARγ, identified in three unrelated subjects with partial lipodystrophy, severe insulin resistance, dyslipidaemia and hypertension are described. Their functional characterization suggests that they inhibit WT action via a novel, non DNA-binding interference mechanism

Variability of DA/DAPI and C Heterochromatic Regions: A Population Study

DA/DAPI and C chromosomal heteromorphic sites (lq, 9q, 15p, 16q, Yq) in a sample of 136 unrelated individuals from the Garfagnana valley (Tuscany, Italy) have been analyzed quantitatively and qualitatively. The variations in length, between homolog heteromorphisms, and intensity of fluorescence of the heterochromatic bands have been compared in two subsamples of the population (upper and middle valley) individualized according to geodemographic criteria. DA/DAPI heterochromatin differed significantly from C heterochromatin, showing a lower average amount and a higher variability at each site. This suggests a differential staining of DNA of the two banding systems. Furthermore, DA/DAPI heterochromatin was less uniformly distributed in the population than C heterochromatin and the regions 16q and Yq discriminated better between subsamples. The variations of DA/DAPI fluorescence at the 15p site demonstrated an excess of homomorphic individuals in the upper valley, which could be related to the mating structure of the population living in this area