The Pathogenic TSH β-Subunit Variant C105Vfs114X Causes a Modified Signaling Profile at TSHR

1) Background: Central congenital hypothyroidism (CCH) is a rare endocrine disorder that can be caused by mutations in the β-subunit of thyrotropin (TSHB). The TSHB mutation C105Vfs114X leads to isolated thyroid-stimulating-hormone-(TSH)-deficiency and results in a severe phenotype. The aim of this study was to gain more insight into the underlying molecular mechanism and the functional effects of this mutation based on two assumptions: a) the three-dimensional (3D) structure of TSH should be modified with the C105V substitution, and/or b) whether the C-terminal modifications lead to signaling differences. 2) Methods: wild-type (WT) and different mutants of hTSH were generated in human embryonic kidney 293 cells (HEK293 cells) and TSH preparations were used to stimulate thyrotropin receptor (TSHR) stably transfected into follicular thyroid cancer cells (FTC133-TSHR cells) and transiently transfected into HEK293 cells. Functional characterization was performed by determination of Gs, mitogen activated protein kinase (MAPK) and Gq/11 activation. 3) Results: The patient mutation C105Vfs114X and further designed TSH mutants diminished cyclic adenosine monophosphate (cAMP) signaling activity. Surprisingly, MAPK signaling for all mutants was comparable to WT, while none of the mutants induced PLC activation. 4) Conclusion: We characterized the patient mutation C105Vfs114X concerning different signaling pathways. We identified a strong decrease of cAMP signaling induction and speculate that this could, in combination with diverse signaling regarding the other pathways, accounting for the patient's severe phenotype

Klinische und molekularbiologische Effekte der Mutation C105Vfs114X im TSHB Gen

Kongenitale Hypothyreosen sind mit einer Inzidenz von etwa 1:3.500 die häufigste angeborene endokrine Erkrankung. Die meisten Patienten weisen eine primäre Hypothyreose auf, die in der Regel durch eine abnorme Entwicklung der Schilddrüse bedingt ist. Sekundäre beziehungsweise zentrale Hypothyreosen sind mit einer Inzidenz von ca. 1:50.000 wesentlich seltener. Eine der Ursachen dafür sind Mutationen im TSHB Gen, welche zu einem isolierten TSH-Mangel führen. Man ging lange davon aus, dass sekundäre Hypothyreosen zu einem wesentlich milderen Phänotyp als primäre Hypothyreosen führen. Dies trifft jedoch nicht für Patienten mit einer Mutation in der β-Untereinheit des TSH und insbesondere nicht für Patienten mit der Mutation C105Vfs114X zu. Hinzu kommt, dass diese Patienten nicht im Neugeborenscreening detektiert werden, da dieses auf der alleinigen Messung von TSH beruht, welches in diesen Fällen in der Regel unterhalb der Nachweisgrenze oder im unteren Normbereich liegt. Patienten mit dieser Mutation fallen mit ausgeprägten Symptomen einer schweren konnatalen Hypothyreose auf und haben langfristige psychomotorische sowie neurokognitive Defizite, da die Mutation meist nicht so früh erkannt wird, dass die Substitutionstherapie mit L-Thyroxin Langzeitschäden verhindert. Weshalb diese Mutation zu einem so schweren Phänotyp führt, sollte in dieser Arbeit untersucht werden. Ein besonderer Pathomechanismus, z.B. im Sinne eines invers-agonistischen Effekts des mutierten TSH am TSH-Rezeptor, wurde seit Ende der 90er Jahren vermutet. Besonderes Augenmerk wurde in dieser Arbeit auf den Verlust einer Disulfidbrückenbindung und den verkürzten C-Terminus gelegt. Dadurch potentiell bedingte Veränderungen der Formation des Proteins könnten diesen spefizischen Pathomechanismus bedingen. Um dies zu untersuchen, wurden in dieser Arbeit verschiedene TSHB-Mutanten generiert und rekombinantes hTSH durch transiente Transfektion in HEK293 Zellen hergestellt. Die Menge des hergestellten hTSH wurde gemessen und das hTSH funktionell charakterisiert. In dieser Arbeit konnte ein invers-agonistischer Effekt der Mutation ausgeschlossen werden. Darüberhinaus zeigten die Experimente, dass der verkürzte C-Terminus nicht ursächlich für die Effekte der Mutation verantwortlich ist. Es ist nicht gelungen eine andere molekulare Ursache für diesen schwerwiegenden Phänotyp zu ermitteln. Dafür bedarf es weiterführender Experimente mit zusätzlichen Mutanten, um weitere Erkenntnisse zu den Auswirkungen dieser Mutation zu erlangen. Ein tieferes Verständnis dieser Mutation könnte nicht nur dazu beitragen, auch Patienten und ihren Eltern das Krankheitsbild schlüssig zu erklären, sondern ggf. auch dazu beitragen, neue zielgerichtete Therapien z.B. für Patienten mit Hyperthyreose zu entwickeln. Nicht zuletzt kann eine auf molekularer Ebene gesicherte Erkenntnis einer schweren Beeinträchtigung der Schilddrüsenhormonbiosynthese dazu beitragen, dass die Strategie des Neugeborenenscreenings auf angeborene Hypothyreose revidiert wird.Congenital hypothyroidism is – with an incidence of approximately 1:3,500 – the most common congenital endocrine disorder. Most patients are diagnosed with primary congenital hypothyroidism, which is in most cases caused by a developmental defect of the thyroid gland. Comparably, secondary (central hypothyroidism) is very rare with an incidence of 1:50,000. One of the reasons for central hypothyroidism are mutations in the TSHB gene, which lead to an isolated TSH-deficiency. It has long been assumed that central hypothyroidism leads to a milder phenotype than primary hypothyroidism does. However, this does not apply to mutations in the β-subunit, especially not to the mutation C105Vfs114X. Furthermore, these patients are not detected through newborn screening as this screening is based on TSH, in which β-subunit mutations result in TSH concentrations below or at the lower end of the reference range. Affected patients therefore present with severe signs of congenital hypothyroidism and with long-term psychomotor and neurocognitive deficiencies if the diagnosis is not made in the neonatal period and the start of l-thyroxine replacement is delayed. The aim of this study was to investigate the mechanism by which this specific mutation leads to a more severe phenotype than other forms of central hypothyroidism. Since the 1990s it was presumed that an inverse-agonistic effect of the mutated TSH at the receptor could cause this phenotype. This again, could be caused by the shortened c-terminus, an altered amino acid sequence or the loss of one of the disulfide bridges, which could lead to a modified formation of the protein. Different mutants were generated for the experiments; recombinant hTSH was produced using transient transfection in HEK293 cells. The produced hTSH was measured and a functional characterization of the wildtype as well as of the mutants was conducted. With these experiments an inverse-agonistic effect of the mutation was ruled out and it was proved that the shortened c-terminus is not responsible for the effects of the mutation. However, it was not possible to identify the exact molecular mechanism. In order to do so, further experiments with additional variants will be required. Besides an explanation for patients and their families, a deeper understanding of this mutation could help to develop novel targeted therapies especially for patients with hyperthyroidism. Not least, molecular evidence for a severely altered production of thyroid hormones would underline the necessity to revise the strategy of newborn screening programs for congenital hypothyroidism

Hepatocyte-specific NRF2 activation controls fibrogenesis and carcinogenesis in steatohepatitis

Background & Aims: In chronic liver diseases, inflammation induces oxidative stress and thus may contribute to the progression of liver injury, fibrosis, and carcinogenesis. The KEAP1/NRF2 axis is a major regulator of cellular redox balance. In the present study, we investigated whether the KEAP1/NRF2 system is involved in liver disease progression in humans and mice. Methods: The clinical relevance of oxidative stress was investigated by liver RNA sequencing in a well-characterized cohort of patients with non-alcoholic fatty liver disease (n = 63) and correlated with histological and clinical parameters. For functional analysis, hepatocyte-specific Nemo knockout (NEMOΔhepa) mice were crossed with hepatocyte-specific Keap1 knockout (KEAP1Δhepa) mice. Results: Immunohistochemical analysis of human liver sections showed increased oxidative stress and high NRF2 expression in patients with chronic liver disease. RNA sequencing of liver samples in a human pediatric NAFLD cohort revealed a significant increase of NRF2 activation correlating with the grade of inflammation, but not with the grade of steatosis, which could be confirmed in a second adult NASH cohort. In mice, microarray analysis revealed that Keap1 deletion induces NRF2 target genes involved in glutathione metabolism and xenobiotic stress (e.g., Nqo1). Furthermore, deficiency of one of the most important antioxidants, glutathione (GSH), in NEMOΔhepa livers was rescued after deleting Keap1. As a consequence, NEMOΔhepa/KEAP1Δhepa livers showed reduced apoptosis compared to NEMOΔhepa livers as well as a dramatic downregulation of genes involved in cell cycle regulation and DNA replication. Consequently, NEMOΔhepa/KEAP1Δhepa compared to NEMOΔhepa livers displayed decreased fibrogenesis, lower tumor incidence, reduced tumor number, and decreased tumor size. Conclusions: NRF2 activation in patients with non-alcoholic steatohepatitis correlates with the grade of inflammation, but not steatosis. Functional analysis in mice demonstrated that NRF2 activation in chronic liver disease is protective by ameliorating fibrogenesis, initiation and progression of hepatocellular carcinogenesis. Lay summary: The KEAP1 (Kelch-like ECH-associated protein-1)/NRF2 (erythroid 2-related factor 2) axis has a major role in regulating cellular redox balance. Herein, we show that NRF2 activity correlates with the grade of inflammation in patients with non-alcoholic steatohepatitis. Functional studies in mice actually show that NRF2 activation, resulting from KEAP1 deletion, protects against fibrosis and cancer.</p