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Treatment of dysferlinopathy with deflazacort: a double-blind, placebo-controlled clinical trial
(2013)
Background: Dysferlinopathies are autosomal recessive disorders caused by mutations in the dysferlin (DYSF) gene encoding the dysferlin protein. DYSF mutations lead to a wide range of muscular phenotypes, with the most prominent being Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B).
Methods: We assessed the one-year-natural course of dysferlinopathy, and the safety and efficacy of deflazacort treatment in a double-blind, placebo-controlled cross-over trial. After one year of natural course without intervention, 25 patients with genetically defined dysferlinopathy were randomized to receive deflazacort and placebo for six months each (1 mg/kg/day in month one, 1 mg/kg every 2nd day during months two to six) in one of two treatment sequences. Results: During one year of natural course, muscle strength declined about 2% as measured by CIDD (Clinical Investigation of Duchenne Dystrophy) score, and 76 Newton as measured by hand-held dynamometry. Deflazacort did not improve muscle strength. In contrast, there is a trend of worsening muscle strength under deflazacort treatment, which recovers after discontinuation of the study drug. During deflazacort treatment, patients showed a broad spectrum of steroid side effects.
Conclusion: Deflazacort is not an effective therapy for dysferlinopathies, and off-label use is not warranted. This is an important finding, since steroid treatment should not be administered in patients with dysferlinopathy, who may be often misdiagnosed as polymyositis.
Das Nebennierenrindenkarzinom (NNR-Ca) gehört mit einer Inzidenz von 1-2/1000000 zu den seltenen malignen Neubildungen. Neben Sarkomen, Hirntumoren, Brustkrebs und Leukämie gehört das NNR-Ca zu den Kerntumoren, durch die das selten vorkommende autosomal dominante Tumor-Prädispositions Syndrom, das Li Fraumeni Syndrom (LFS) gekennzeichnet ist. Das LFS wird mit Keimbahnmutationen im Tumorsuppressor Gen TP53 in Verbindung gebracht. Die vorliegende Arbeit untersucht TP53 Keimbahnmutationen und -polymorphismen und ihre Auswirkung auf klinische Faktoren bei einem großen Kollektiv von erwachsenen NNR-Ca Patienten.
Es wurde DNS aus Blut und teilweise aus Tumorgewebe von Patienten aus dem Deutschen Nebennierenrindenkarzinom Register extrahiert und die Exons 2 bis 11 von TP53 sequenziert. Darüber hinaus wurde der Nachweis der Mutationen und eines Loss of Heterozgosity von TP53 im Tumorgewebe und die immunhistochemische Färbung von p53 vorgenommen. Die anschließende Auswertung der Daten erfolgte unter Einbeziehung des klinischen Verlaufs der Krankheit bei den Patienten.
In dieser Arbeit konnten vier NNR-Ca Patienten (3,9 %) mit mindestens einer Keimbahnmutation im TP53 identifiziert werden, bei den unter 40-jährigen entspricht dies einem Anteil von 13,0 %. Unter der Altersgrenze von 40 Jahren sollte daher ein TP53 Mutationsscreening erwogen werden.
Die Auswertung der Polymorphismen zeigte, dass diese einen Einfluss auf die Entstehung und den klinischen Verlauf des NNR-Cas zu haben scheinen, jedoch weitere Studien nötig sind.
The IL22RA2 locus is associated with risk for multiple sclerosis (MS) but causative variants are yet to be determined. In a single nucleotide polymorphism (SNP) screen of this locus in a Basque population, rs28385692, a rare coding variant substituting Leu for Pro at position 16 emerged significantly (p = 0.02). This variant is located in the signal peptide (SP) shared by the three secreted protein isoforms produced by IL22RA2 (IL-22 binding protein-1(IL-22BPi1), IL-22BPi2 and IL-22BPi3). Genotyping was extended to a Europe-wide case-control dataset and yielded high significance in the full dataset (p = 3.17 × 10\(^{-4}\)). Importantly, logistic regression analyses conditioning on the main known MS-associated SNP at this locus, rs17066096, revealed that this association was independent from the primary association signal in the full case-control dataset. In silico analysis predicted both disruption of the alpha helix of the H-region of the SP and decreased hydrophobicity of this region, ultimately affecting the SP cleavage site. We tested the effect of the p.Leu16Pro variant on the secretion of IL-22BPi1, IL-22BPi2 and IL-22BPi3 and observed that the Pro16 risk allele significantly lowers secretion levels of each of the isoforms to around 50%–60% in comparison to the Leu16 reference allele. Thus, our study suggests that genetically coded decreased levels of IL-22BP isoforms are associated with augmented risk for MS.
Fanconi-Anämie gehört zu den Chromosomenbruch-Syndromen und zeichnet sich durch eine genomische Instabilität aus. Die genomische Instabilität ist auch Ursache häufiger Rückmutationen. Solche kommen vermehrt in Genabschnitten hoher Sequenzvariabilität vor. Insbesondere gilt dies für das Exon 10 des FANCA-Gens. Dieser Abschnitt des Gens wurde herausgesucht, da dieser als hypermutabler Bereich in der Literatur beschrieben ist. In dieser Arbeit sollte untersucht werden, inwieweit Sequenzen des Exon 10 somatische Instabilität aufweisen. Hierzu wurden Fibroblasten eines Patienten und entsprechende Kontrollen in der Zellkultur seriell passagiert und gealtert. Im Zuge der Zellalterung konnte gezeigt werden, dass sich die Wachstumsrate verringerte. Die Zellen, die mit MMC behandelt wurden, stellten das Wachstum nach 1-2 Zellpassagen ein. Der Sequenzabschnitt aus FANCA wurde aus isolierter DNA der Zellen amplifiziert und im PCR-TOPO TA kloniert. Die erhaltenen Klone wurden sequenziert. Die Sequenzanalysen ergaben als häufigste Basensubstitution einen T nach C Austausch oder revers komplementär einen Austausch von A nach G. Dies wird als Artefakt interpretiert. Vermutliche Ursache ist die relativ hohe Fehlerrate der Taq-Polymerase. Für die Amplifizierung des Genomabschnittes aus dem FANCA-Gen sollte eine Polymerase verwendet werden, die eine höhere Genauigkeit als die Taq-Polymerase aufweist, wie z. B. die Pfu-Polymerase.
After the recent emergence of SARS-CoV-2 infection, unanswered questions remain related to its evolutionary history, path of transmission or divergence and role of recombination. There is emerging evidence on amino acid substitutions occurring in key residues of the receptor-binding domain of the spike glycoprotein in coronavirus isolates from bat and pangolins. In this article, we summarize our current knowledge on the origin of SARS-CoV-2. We also analyze the host ACE2-interacting residues of the receptor-binding domain of spike glycoprotein in SARS-CoV-2 isolates from bats, and compare it to pangolin SARS-CoV-2 isolates collected from Guangdong province (GD Pangolin-CoV) and Guangxi autonomous regions (GX Pangolin-CoV) of South China. Based on our comparative analysis, we support the view that the Guangdong Pangolins are the intermediate hosts that adapted the SARS-CoV-2 and represented a significant evolutionary link in the path of transmission of SARS-CoV-2 virus. We also discuss the role of intermediate hosts in the origin of Omicron.
PTH1R Mutants Found in Patients with Primary Failure of Tooth Eruption Disrupt G-Protein Signaling
(2016)
Aim
Primary failure of tooth eruption (PFE) is causally linked to heterozygous mutations of the parathyroid hormone receptor (PTH1R) gene. The mutants described so far lead to exchange of amino acids or truncation of the protein that may result in structural changes of the expressed PTH1R. However, functional effects of these mutations have not been investigated yet.
Materials and Methods
In HEK293 cells, PTH1R wild type was co-transfected with selected PTH1R mutants identified in patients with PFE. The effects on activation of PTH-regulated intracellular signaling pathways were analyzed by ELISA and Western immunoblotting. Differential effects of wild type and mutated PTH1R on TRESK ion channel regulation were analyzed by electrophysiological recordings in Xenopus laevis oocytes.
Results
In HEK293 cells, activation of PTH1R wild type increases cAMP and in response activates cAMP-stimulated protein kinase as detected by phosphorylation of the vasodilator stimulated phosphoprotein (VASP). In contrast, the PTH1R mutants are functionally inactive and mutant PTH1R/Gly452Glu has a dominant negative effect on the signaling of PTH1R wild type. Confocal imaging revealed that wild type PTH1R is expressed on the cell surface, whereas PTH1R/Gly452Glu mutant is mostly retained inside the cell. Furthermore, in contrast to wild type PTH1R which substantially augmented K+ currents of TRESK channels, coupling of mutated PTH1R to TRESK channels was completely abolished.
Conclusions
PTH1R mutations affect intracellular PTH-regulated signaling in vitro. In patients with primary failure of tooth eruption defective signaling of PTH1R mutations is suggested to occur in dento-alveolar cells and thus may lead to impaired tooth movement.
Basing on the assumption that frontotemporal lobar degeneration (FTLD), schizophrenia and bipolar disorder (BPD) might share common aetiological mechanisms, we analyzed genetic variation in the FTLD risk gene progranulin (GRN) in a German population of patients with schizophrenia (n=271) or BPD (n=237) as compared with 574 age-, gender-and ethnicity-matched controls. Furthermore, we measured plasma progranulin levels in 26 German BPD patients as well as in 61 Italian BPD patients and 29 matched controls. A significantly decreased allelic frequency of the minor versus the wild-type allele was observed for rs2879096 (23.2 versus 34.2%, P<0.001, OR: 0.63, 95% CI: 0.49-0.80), rs4792938 (30.7 versus 39.7%, P=0.005, OR: 0.70, 95% CI: 0.55-0.89) and rs5848 (30.3 versus 36.8, P=0.007, OR: 0.71, 95% CI: 0.56-0.91). Mean +/- SEM progranulin plasma levels were significantly decreased in BPD patients, either Germans or Italians, as compared with controls (89.69 +/- 3.97 and 116.14 +/- 5.80 ng/ml, respectively, versus 180.81 +/- 18.39 ng/ml P<0.001) and were not correlated with age. In conclusion, GRN variability decreases the risk to develop BPD and schizophrenia, and progranulin plasma levels are significantly lower in BPD patients than in controls. Nevertheless, a larger replication analysis would be needed to confirm these preliminary results.
Two brothers in their fifties presented with a medical history of suspected fungal allergy, allergic bronchopulmonary aspergillosis, alveolitis, and invasive aspergillosis and pulmonary fistula, respectively. Eventually, after a delay of 50 years, chronic granulomatous disease (CGD) was diagnosed in the index patient. We found a new splice mutation in the NCF2 (p67-phox) gene, c.1000+2T -> G, that led to several splice products one of which lacked exons 11 and 12. This deletion was in frame and allowed for remarkable residual NADPH oxidase activity as determined by transduction experiments using a retroviral vector. We conclude that p67-phox which lacks the 34 amino acids encoded by the two exons can still exert considerable functional activity. This activity can partially explain the long-term survival of the patients without adequate diagnosis and treatment, but could not prevent progressing lung damage.
In der vorliegenden Arbeit wurden aus den Testzentren Würzburg und Wien DNA-Proben von Patienten, die im Laufe einer Anästhesie eine maligne Hyperthermie (MH) entwickelt hatten, und deren Angehörigen auf MH-verursachende Mutationen im Gen für den Ryanodinrezeptor 1 (RYR 1) untersucht. Es wurde dabei eine Hotspotregion des RYR 1 ausgewählt, für die bereits im Vorfeld mehrere Mutationen bekannt waren. Das Screening wurde mit Hilfe der Methode single-stranded conformation polymorphism (SSCP) durchgeführt. Eine dem abweichenden Laufverhalten eventuell zugrunde-liegende Mutation wurde anschließend durch die automatische Sequenzierung identifiziert. Unter 190 Patienten aus 126 Familien konnte in 18 Fällen eine Mutation gefunden werden. Das entspricht einer Detektionsrate von 14,29%. Insgesamt traten 10 verschiedene Mutationen auf, von denen eine (G6377A) vorher noch nicht beschrieben war. Die Mutationshäufigkeiten unterschieden sich zum Teil erheblich innerhalb der beiden untersuchten Populationen und im Vergleich zu Ergebnissen anderer Arbeitsgruppen. Alle Indexpatienten und viele Angehörige hatten sich bereits einem in-vitro-Kontrakturtest (IVCT) zur Diagnostik der MH unterzogen. So konnten die Ergebnisse des IVCT mit denen der genetischen Untersuchung verglichen werden. Es fand sich eine gute Übereinstimmung, die die Zuverlässigkeit des IVCT stützt. Begleitend zur Screeninguntersuchung wurden die Methoden SSCP und automatische Sequenzierung hinsichtlich ihrer Anwendbarkeit im Screening großer Patientenkollektive bewertet. Bei vergleichbarer Sicherheit ist mit dem SSCP in kürzerer Zeit bei geringerem Kostenaufwand eine größere Anzahl an Patientenproben auswertbar. Der Stellenwert der genetischen Diagnostik bei der MH wurde als ideale Methode zur Identifikation betroffener Familienmitglieder in MH-Familien mit bekannter Mutation bestätigt.
Lack of Ubiquitin Specific Protease 8 (USP8) Mutations in Canine Corticotroph Pituitary Adenomas
(2016)
Purpose
Cushing’s disease (CD), also known as pituitary-dependent hyperadrenocorticism, is caused by adrenocorticotropic hormone (ACTH)-secreting pituitary tumours. Affected humans and dogs have similar clinical manifestations, however, the incidence of the canine disease is thousand-fold higher. This makes the dog an obvious model for studying the pathogenesis of pituitary-dependent hyperadrenocorticism. Despite certain similarities identified at the molecular level, the question still remains whether the two species have a shared oncogenetic background. Recently, hotspot recurrent mutations in the gene encoding for ubiquitin specific protease 8 (USP8) have been identified as the main driver behind the formation of ACTH-secreting pituitary adenomas in humans. In this study, we aimed to verify whether USP8 mutations also play a role in the development of such tumours in dogs.
Methods
Presence of USP8 mutations was analysed by Sanger and PCR-cloning sequencing in 38 canine ACTH-secreting adenomas. Furthermore, the role of USP8 and EGFR protein expression was assessed by immunohistochemistry in a subset of 25 adenomas.
Results
None of the analysed canine ACTH-secreting adenomas presented mutations in the USP8 gene. In a subset of these adenomas, however, we observed an increased nuclear expression of USP8, a phenotype characteristic for the USP8 mutated human tumours, that correlated with smaller tumour size but elevated ACTH production in those tumours.
Conclusions
Canine ACTH-secreting pituitary adenomas lack mutations in the USP8 gene suggesting a different genetic background of pituitary tumourigenesis in dogs. However, elevated nuclear USP8 protein expression in a subset of tumours was associated with a similar phenotype as in their human counterparts, indicating a possible end-point convergence of the different genetic backgrounds in the two species. In order to establish the dog as a useful animal model for the study of CD, further comprehensive studies are needed.