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

Mutations in thyroid hormone receptor α1 cause premature neurogenesis and progenitor cell depletion in human cortical development.

Mutations in the thyroid hormone receptor α 1 gene (THRA) have recently been identified as a cause of intellectual deficit in humans. Patients present with structural abnormalities including microencephaly, reduced cerebellar volume and decreased axonal density. Here, we show that directed differentiation of THRA mutant patient-derived induced pluripotent stem cells to forebrain neural progenitors is markedly reduced, but mutant progenitor cells can generate deep and upper cortical layer neurons and form functional neuronal networks. Quantitative lineage tracing shows that THRA mutation-containing progenitor cells exit the cell cycle prematurely, resulting in reduced clonal output. Using a micropatterned chip assay, we find that spatial self-organization of mutation-containing progenitor cells in vitro is impaired, consistent with down-regulated expression of cell-cell adhesion genes. These results reveal that thyroid hormone receptor α1 is required for normal neural progenitor cell proliferation in human cerebral cortical development. They also exemplify quantitative approaches for studying neurodevelopmental disorders using patient-derived cells in vitro.NIHR Cambridge Biomedical Centr

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

Correlation of mutations in the CFTR gene with atypical CF diseases

Cystic fibrosis (CF) is the most common autosomal recessive disease in Caucasians. It is caused by defects in the CFTR gene which encodes the cystic fibrosis transmembrane regulator (CFTR) protein. The aim of this study was to evaluate the frequency and nature of mutations in the CFTR gene in atypical CF diseases and specifically a) male infertility, in 53 males with congenital bilateral absence of the vas deferens (CBAVD), 42 males with obstructive azoospermia without CBAVD and 75 males with severe oligozoospermia and b) chronic pulmonary diseases of unknown origin, in 53 patients with disseminated bronchiectasis,34 patients with asthma and 13 patients with chronic obstructive pulmonary disease (COPD), and compare the results to a group of 208 healthy subjects. The entire coding region of the CFTR gene was analyzed using denaturing gradient gel electrophoresis (DGGE), for the detection of mutations and polymorphisms. The intron 8 poly-thymidine tract (IVS8-Tn) and the number of TG repeats in the same intron (IVS8-TGm) which determine the proportion of normal CFTR transcripts containing exon 9 and producing functional CFTR protein, were also analyzed. Additionally we studied the CFTR mRNA extracted from nasal epithelial cells in four splicing mutations (c.621+3A>G, c.711+3A>G, IVS8-5T, p.D565G) and one polymorphism (c.2752-15C/G).In the male infertility group a total of 36 different molecular defects were identified including five novel mutations (p.D565G, c.2790-8C/G, p.I521F, p.G817V, p.C491S) in 56 males. More specifically 62.3% (33/53) (p0.1) of males with oligozoospermia had at least one CFTR mutation, compared to 14.9% of the control subjects (31/208). In the chronic pulmonary group a total of 23 different molecular defects were identified including four novel mutations (p.T388M, p.M1R, p.V11I, p.Q378R) in 28 patients. More specifically 38.2% (13/34) (p0.1)) of patients with disseminated bronchiectasis had at least one CFTR mutation and 30.8% (4/13) (pG, c.711+3A>G and D565G lead to the production of aberrantly spliced CFTR transcripts. They cause reduction in the amount of normal CFTR mRNA and reduction in the amount of functional CFTR protein, so that they affect the phenotypic CF variability and modulate the disease severity.Η Κυστική Ίνωση αποτελεί το συχνότερο κληρονομικό νόσημα στη λευκή φυλή που κληρονομείται με τον υπολειπόμενο αυτοσωμικό χαρακτήρα. Προκαλείται από μεταλλάξεις στο CFTR γονίδιο που κωδικοποιεί την CFTR πρωτεΐνη (Cystic Fibrosis Transmembrane Regulator). Σκοπός της παρούσας μελέτης είναι η συσχέτιση των μοριακών διαταραχών της κυστικής ίνωσης με άτυπες κλινικές μορφές της νόσου και συγκεκριμένα α) την ανδρική υπογονιμότητα, σε 53 άρρενες με συγγενή αμφοτερόπλευρη έλλειψη σπερματικού πόρου (CBAVD), 42 άρρενες με αποφρακτική αζωοσπερμία χωρίς έλλειψη των σπερματικών πόρων και 75 άρρενες με σοβαρή ολιγοσπερμία και β) τις χρόνιες πνευμονοπάθειες αδιευκρίνιστης αιτιολογίας, σε 53 ασθενείς με διάχυτες βρογχιεκτασίες, 34 ασθενείς με άσθμα και 13 ασθενείς με χρόνια αποφρακτική πνευμονοπάθεια, και να συγκρίνει τα αποτελέσματα με τα αντίστοιχα από μια ομάδα 208 υγιών ατόμων. Έγινε ανάλυση όλων των κωδικοποιουσών περιοχών του CFTR γονιδίου, χρησιμοποιώντας τη μέθοδο της ηλεκτροφόρησης σε πήκτωμα με κλίση αποδιατακτικού (DGGE), για την ανίχνευση μεταλλάξεων και πολυμορφισμών. Επίσης μελετήθηκαν η πολυθυμιδική αλληλουχία στο ιντρόνιο 8 (IVS8-Tn) και ο αριθμός των TG επαναλήψεων στο ίδιο ιντρόνιο (IVS8-TGm), τα οποία καθορίζουν το ποσοστό των φυσιολογικών CFTR μεταγράφων που περιέχουν το εξόνιο 9 και παράγουν λειτουργική CFTR πρωτεΐνη.Επιπροσθέτως έγινε ανάλυση των CFTR μεταγράφων (mRNA) σε συγκεκριμένες μοριακές διαταραχές που απαντούν χαρακτηριστικά στους ασθενείς με άτυπες κλινικές μορφές κυστικής ίνωσης, και συγκεκριμένα 4 μεταλλάξεις ματίσματος ( c.621+3A>G, c.711+3A>G, IVS8-5T, p.D565G) και 1 πολυμορφική θέση (c.2752-15C/G), χρησιμοποιώντας ως υλικό επιθηλιακά κύτταρα του ρινικού βλεννογόνου.Στους άνδρες με υπογονιμότητα ανευρέθησαν συνολικά 36 διαφορετικές μοριακές διαταραχές, συμπεριλαμβανομένων πέντε νέων μεταλλάξεων (p.D565G, c.2790-8C/G, p.I521F, p.G817V και p.C491S) σε 56 άνδρες. Συγκεκριμένα στην ομάδα με CBAVD το ποσοστό των ανδρών με μία τουλάχιστον μοριακή διαταραχή στο γονίδιο της κυστικής ίνωσης ανέρχεται σε 62.3% (33/53) (p0.1), σε σύγκριση με την ομάδα των φυσιολογικών μαρτύρων όπου το αντίστοιχο ποσοστό ήταν 14.9% (31/208). Στους ασθενείς με χρόνια πνευμονοπάθεια ανευρέθησαν συνολικά 23 διαφορετικές μοριακές διαταραχές, συμπεριλαμβανομένων τεσσάρων νέων μεταλλάξεων (p.T388M, p.M1R, p.V11I, p.Q378R) σε 28 ασθενείς. Συγκεκριμένα στην ομάδα με άσθμα το ποσοστό των ασθενών με μία τουλάχιστον μοριακή διαταραχή στο γονίδιο της κυστικής ίνωσης ανέρχεται σε 38.2% (13/34) (p0.1) και στην ομάδα με χρόνια αποφρακτική πνευμονοπάθεια σε 30.8% (4/13) (pG, c.711+3A>G και p.D565G οδηγούν στην παραγωγή εναλλακτικών παθολογικών CFTR μεταγράφων. Προκαλούν μειωμένα ποσά φυσιολογικού CFTR mRNA και κατά συνέπεια μειωμένα ποσά λειτουργικής CFTR πρωτεΐνης, με τελικό αποτέλεσμα να επηρεάζουν ποικιλοτρόπως τον φαινότυπο των ασθενών και να τροποποιούν τη βαρύτητα της κυστικής ίνωσης

Myostatin serum levels in children with type 1 diabetes mellitus

Purpose Type 1 diabetes mellitus (T1DM) can cause several complications, among them myopathy, which can appear even in adolescents. This is of importance, since skeletal muscle is the largest of the insulin-sensitive tissues and thus plays a significant role in glucose homeostasis. A prime regulator of skeletal muscle mass is myostatin, a protein which has a negative role in skeletal muscle development but also in glucose homeostasis, causing insulin resistance. Since myopathy is a complication of T1DM and myostatin is a fundamental regulator of skeletal muscle and is also involved in glucose homeostasis, we investigated the serum levels of myostatin in children with T1DM. Methods We determined myostatin serum levels using ELISA in 87 children with T1DM aged 10.62 +/- 3.94 years, and in 75 healthy children aged 10.46 +/- 3.32 years old. Results My omicron statin was significantly elevated in T1DM compared to the healthy control children (23.60 +/- 7.70 vs 16.74 +/- 6.95 ng/ml, p < 0.0001). Myostatin was not correlated with body mass index (BMI) SD or hemoglobin A1c (HbA1c). Conclusion Children with T1DM have significantly higher serum levels of myostatin compared to healthy children of the same age and BMI SD. The elevated myostatin in T1DM could reflect impaired muscle function and/or glucose metabolism, or could represent a homeostatic mechanism

Evaluation of the Performance of 18F-Fluorothymidine Positron Emission Tomography/Computed Tomography (18F-FLT-PET/CT) in Metastatic Brain Lesions

18F-fluorothymidine (18F-FLT) is a radiolabeled thymidine analog that has been reported to help monitor tumor proliferation and has been studied in primary brain tumors; however, knowledge about 18F-FLT positron emission tomography/computed tomography (PET/CT) in metastatic brain lesions is limited. The purpose of this study is to evaluate the performance of 18F-FLT-PET/CT in metastatic brain lesions. A total of 20 PET/CT examinations (33 lesions) were included in the study. Semiquantitative analysis was performed: standard uptake value (SUV) with the utilization of SUVmax, tumor-to-background ratio (T/B), SUVpeak, SUV1cm3, SUV0.5cm3, SUV50%, SUV75%, PV50% (volume × SUV50%), and PV75% (volume × SUV75%) were calculated. Sensitivity, specificity, and accuracy for each parameter were calculated. Optimal cutoff values for each parameter were obtained. Using a receiver operating characteristic (ROC) curve analysis, the optimal cutoff values of SUVmax, T/B, and SUVpeak for discriminating active from non-active lesions were found to be 0.615, 4.21, and 0.425, respectively. In an ROC curve analysis, the area under the curve (AUC) is higher for SUVmax (p-value 0.017) compared to the rest of the parameters, while using optimal cutoff T/B shows the highest sensitivity and accuracy. PVs (proliferation × volumes) did not show any significance in discriminating positive from negative lesions. 18F-FLT-PET/CT can detect active metastatic brain lesions and may be used as a complementary tool. Further investigation should be performed