Mutation in the mouse histone gene Hist2h3c1 leads to degeneration of the lens vesicle and severe microphthalmia

During an ENU (N-ethyl-N-nitrosourea) mutagenesis screen, we observed a dominant small-eye mutant mouse with viable homozygotes. A corresponding mutant line was established and referred to as Aey69 (abnormality of the eye #69). Comprehensive phenotyping of the homozygous Aey69 mutants in the German Mouse Clinic revealed only a subset of statistically significant alterations between wild types and homozygous mutants. The mutation causes microphthalmia without a lens but with retinal hyperproliferation. Linkage was demonstrated to mouse chromosome 3 between the markers D3Mit188 and D3Mit11. Sequencing revealed a 358 A- > C mutation (I1e120Leu) in the Hist2h3c1 gene and a 71 T- > C (Val24Ala) mutation in the Gja8 gene. Detailed analysis of eye development in the homozygous mutant mice documented a perturbed lens development starting -from the lens vesicle stage including decreasing expression of crystallins as well as of lens-specific transcription - factors like PITX3 and FOXE3. In contrast, we observed an early expression of retinal progenitor cells characterized by several markers including BRN3 (retinal ganglion cells) and OTX2 (cone photoreceptors). The changes in the retina at the early embryonic stages of E11.5-E15.5 happen in parallel with apoptotic processes in the lens at the respective stages. The excessive retinal hyperproliferation is characterized by an increased level of Ki67. The hyperproliferation, however, does not disrupt the differentiation and appearance of the principal retinal cell types at postnatal stages, even if the overgrowing retina covers finally the entire bulbus of the eye. Morpholino-mediated knock-down of the hist2h3ca1 gene in zebrafish leads to a specific perturbation of lens development. When injected into zebrafish zygotes, only the mutant mouse mRNA leads to severe malformations, ranging from cyclopia to severe microphthalmia. The wild-type Hist2h3c1 mRNA can rescue the morpholino-induced defects corroborating its specific function in lens development. Based upon these data, it is concluded that the ocular function of the Hist2h3c1 gene (encoding a canonical H3.2 variant) is conserved throughout evolution. Moreover, the data highlight also the importance of Hist2h3c1 in the coordinated formation of lens and retina during eye development

Transcript expression-aware annotation improves rare variant interpretation

The acceleration of DNA sequencing in samples from patients and population studies has resulted in extensive catalogues of human genetic variation, but the interpretation of rare genetic variants remains problematic. A notable example of this challenge is the existence of disruptive variants in dosage-sensitive disease genes, even in apparently healthy individuals. Here, by manual curation of putative loss-of-function (pLoF) variants in haploinsufficient disease genes in the Genome Aggregation Database (gnomAD)(1), we show that one explanation for this paradox involves alternative splicing of mRNA, which allows exons of a gene to be expressed at varying levels across different cell types. Currently, no existing annotation tool systematically incorporates information about exon expression into the interpretation of variants. We develop a transcript-level annotation metric known as the 'proportion expressed across transcripts', which quantifies isoform expression for variants. We calculate this metric using 11,706 tissue samples from the Genotype Tissue Expression (GTEx) project(2) and show that it can differentiate between weakly and highly evolutionarily conserved exons, a proxy for functional importance. We demonstrate that expression-based annotation selectively filters 22.8% of falsely annotated pLoF variants found in haploinsufficient disease genes in gnomAD, while removing less than 4% of high-confidence pathogenic variants in the same genes. Finally, we apply our expression filter to the analysis of de novo variants in patients with autism spectrum disorder and intellectual disability or developmental disorders to show that pLoF variants in weakly expressed regions have similar effect sizes to those of synonymous variants, whereas pLoF variants in highly expressed exons are most strongly enriched among cases. Our annotation is fast, flexible and generalizable, making it possible for any variant file to be annotated with any isoform expression dataset, and will be valuable for the genetic diagnosis of rare diseases, the analysis of rare variant burden in complex disorders, and the curation and prioritization of variants in recall-by-genotype studies.Peer reviewe

Genetische und phänotypische Charakterisierung eines Mausmodells für erbliche Polycythämie

Im Rahmen des Münchener ENU-Mausmutagenese-Projekts wurde durch chemische Mutagenese die dominante mutante Mauslinie MVD013 erzeugt. Die Linie wurde an Hand einer erhöhten Zahl und eines verringerten Volumens der Erythrozyten etabliert. Außerdem zeigten einzelne ältere mutante Tiere dieser Linie zusätzliche pathologische Veränderungen wie Darmtumoren und Megakolon. Ziel der Arbeit war es, Untersuchungen zur Identifizierung der ursächlichen Mutation in der Mauslinie MVD013 durchzuführen und die pathologischen Auswirkungen zu charakterisieren, um so ihre Eignung als Tiermodell für Polycythämie und Tumorerkrankungen zu evaluieren. Mittels der durchgeführten Grob- und Feinkartierungen wurde die Lage der ursächlichen Mutation auf Chromosom 5, 59,3-89,7 Mb eingegrenzt. Durch Sequenzanalyse der Kandidatengene Kit, Pdgfra und Kdr konnte keine Mutation im kodierenden Bereich dieser Gene nachgewiesen werden. Die mutante Mauslinie MVD013 wurde im Jahr 2008 im Primärscreen der Deutsche Mausklinik untersucht. Zusammen mit den zusätzlichen sekundären Untersuchungen wurde eine umfassende Phänotypbeschreibung der Mauslinie MVD013 erstellt. Die hämatologische Analyse zeigt ein der humanen Polycythämia vera entsprechendes Blutbild, mit leichter Abweichung vom typischen PV Befund in Richtung Thrombozytopenie. Die klinisch-chemischen Untersuchungen ergaben deutlich erniedrigte Glukosespiegel und Eisenkonzentrationen im Plasma der mutanten Tiere. Die Ergebnisse der Analyse zusätzlicher Parameter des Eisenstoffwechsels und die Ergebnissen des intraperitonealen Glukosetoleranztests deuten auf einen erhöhten Eisen- und Glukoseverbrauch bei mutanten Tieren hin. Anhand der Analyse der Blutgasparameter und der Erythropoetinkonzentration im Serum wurde das Vorliegen einer sekundären Erythrozytose ausgeschlossen. Die Untersuchungen der hämatopoetischen Vorläuferzellen geben einen Hinweis auf myelosuppressive Prozesse im Knochenmark der gealterten mutanten Tiere, was mit Symptomen der humanen Post-Polycythämia vera Myelofibrosis vergleichbar ist. Die mutanten Tiere der Linie MVD013 entwickeln gastrointestinale Stromatumoren in Magen und Darm sowie eine Dilatation (Megaceacum) im Bereich des Blinddarms. Somit kann die dominant mutante Mauslinie MVD013 als ein vielversprechendes Mausmodell für Polycythämia vera, gastrointestinale Stromatumoren und pathologische Veränderungen mit Störungen der Darmperistaltik dienen.MVD013, a mutant mouse line showing a dominantly inherited polycytemia phenotype in the peripheral blood cell count, was established within the Munich ENU mouse mutagenesis project by the Clinical Chemistry Screen. Additionally, gastrointestinal tumors and the development of a Megaceacum were observed in individual aged mutant mice. The aim of this project was to characterizing the mutant phenotype of this mouse line and to conduct investigations to identify the causative mutation, in order to evaluate the usefulness of MVD013 mice as a model of polycythemia and other types of cancer. SNP analysis and additional fine mapping showed the highest possibility of the mutation to be located on chromosome 5 between 59,3 and 89,7 Mb. We sequenced the most promising candidate genes Kdr, Kit and Pdgfra. The latter two genes are associated with gastro-intestinal stromal tumors (GIST) and myeloproliferative disorders. Kdr coding for the vascular endothelial growth factor receptor is essential for the differentiation of hematopoietic precursors and is associated with several kinds of cancers. Our results show that these three genes do not have a mutation in the coding part of the gene and in promoter regions of the Kit or Pdgfra gene. Based on a comprehensive phenotypic characterization of this mouse line in a primary screen conducted 2008 by the German Mouse Clinic, and a variety of additional investigation, a comprehensive description of the mutant phenotype has been generated. Concerning the haematological parameters MVD013 mutant mice show similar symptoms like human patients suffering from Polycythemia vera (PV), with the exception of a platelet count tending towards thrombocytopenia, what is usually not seen in human PV-patients. The results obtained from clinical-chemical plasma analysis indicated decreased plasma glucose and iron levels in mutant animals. Together with the data of the IpGTT and additional measures of further iron-metabolism-related parameters, these results indicate an increased glucose and iron consumption in mutant MVD013 mice. A secondary erythrocytosis due to Erythropoetin-stimulation was excluded by additional analyses such as measurement of erythropoietin level in plasma and blood gas analysis. Further the results suggest an increased iron and glucose consumption in mutant mice, and the development of Gastrointestinal stromal tumors (GIST) in association with an impaired intestinal motility leading to Megaceacum-development. This mouse line might represent a model that can be used to elucidate regulatory mechanisms that playing a role in the development of GISTs, peristaltic dysfunction or polycythemias in humans

Effect of bariatric surgery on liver metabolism -studies using positron emission tomography

Obesity is a significant and increasing health challenge. Obesity is strongly associated with the incidence of type 2 diabetes (T2D). In obese individuals, liver insulin resistance is a major factor in the development and pathophysiology of T2D. Currently, bariatric surgery is the most effective therapy for morbidly obese patients. The aim of this thesis was to study the effects of surgery-induced weight loss on glucose and lipid metabolism in the liver, and to understand the beneficial effects of bariatric surgery on fatty liver, insulin resistance and T2D. Multimodality imaging by positron emission tomography (PET) combined with magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) were utilized to study brain glucose uptake (BGU) and hepatic glucose uptake (HGU), hepatic fatty acid uptake, liver blood flow and liver fat content in morbidly obese patients (n=46) with and without T2D before and six-months after surgery (either sleeve gastrectomy (SG) or Roux-en-Y gastric bypass (RYGB)). The results were then compared to healthy subjects. Preoperatively, insulin-stimulated HGU was lower in obese patients before surgery as compared to the non-obese controls. HGU increased by 30-40% after surgery as compared to the preoperative situation. Postoperatively, liver fat and endogenous glucose production (EGP) were comparable to lean controls. Before surgery, the liver fatty acid uptake was increased in obese subjects as compared to lean controls and associated with body adiposity. Liver fatty acid uptake decreased after surgery but was still high compared to controls. Portal blood flow (per volume of tissue) was increased after surgery, but as the liver volume decreased along with the surgeryinduced weight loss, the whole organ blood flow was unchanged. A positive association between BGU and EGP during insulin stimulation was found in obese subjects and the association persisted after surgery. High BGU at baseline predicted a smaller improvement in fasting plasma glucose at a 2 and 3 year follow-up. The results of this study suggest that bariatric surgery, either SG and RYGB, is effective in improving hepatic glucose metabolism. The persistence of high fatty acid uptake, despite a normal fat content in the liver, suggests a change in the use of fatty acids from storage to oxidation after surgery. Accelerated portal blood flow may relate to improved liver metabolism after surgery. Moreover, these findings suggest the presence of a brain-liver axis in morbidly obese individuals. This axis might contribute to further deterioration of glucose homeostasis.Lihavuusleikkauksen vaikutus maksan aineenvaihduntaan : tutkimuksia positroniemissiotomografiaa käyttäen Lihavuus on merkittävä ja paheneva terveysongelma. Tyypin 2 diabeteksen (T2D) ilmaantuvuus on yhteydessä lihavuuteen. Maksan insuliiniresistenssi vaikuttaa ratkaisevasti T2D:n kehittymiseen lihavilla henkilöillä. Lihavuusleikkaus on nykyisin tehokkain lihavuuden hoitokeino. Väitöskirjan tarkoitus oli tutkia lihavuusleikkauksen jälkeisen painonlaskun vaikutusta maksan glukoosi- ja rasva-aineenvaihduntaan ja ymmärtää lihavuusleikkauksen vaikutusta rasvamaksaan, insuliiniresistenssiin ja T2D:een. Positroniemissiotomografia (PET), magneettiresonanssi (MRI) ja magneettispektroskopia (MRS) -kuvantamisen avulla tutkittiin aivojen ja maksan glukoosin soluunottoa, maksan rasvahappojen soluunottoa, maksan verenkiertoa ja maksan kokoa ja rasvapitoisuutta lihavilla ennen lihavuusleikkausta ja kuusi kuukautta leikkauksen (joko mahalaukun kavennusleikkaus tai mahalaukun ohitusleikkaus) jälkeen. Tutkimuksessa käytettiin terveitä, normaalipainoisia verrokkiryhmänä. Ennen leikkausta maksan glukoosin soluunottokyky oli heikentynyt lihavilla ja se parani 30-40%:lla lihavuusleikkauksen jälkeen leikkausta edeltäneeseen tilanteeseen verrattuna. Leikkauksen jälkeen maksan rasvapitoisuus ja maksan glukoosin tuotanto vähenivät terveiden, normaalipainoisten verrokkien tasolle. Maksan rasvahappojen soluunotto oli lisääntynyt lihavilla ennen leikkausta ja se oli yhteydessä kehon rasvapitoisuuteen. Leikkauksen jälkeen rasvahappojen soluunotto väheni jonkin verran, mutta ei normaalistunut. Maksan porttilaskimon verenvirtaus (ml/min/ml) lisääntyi lihavuusleikkauksen jälkeen, mutta koska maksan koko pieneni leikkauksen jälkeen ei koko elimen verenvirtaus muuttunut. Lihavilla aivojen ja maksan glukoosin soluunotto olivat yhteydessä toisiinsa, ja yhteys säilyi lihavuusleikkauksen jälkeen. Leikkausta edeltänyt lisääntynyt aivojen glukoosin soluunotto ennusti vähäisempää paastoglukoosin laskua 2 ja 3 vuoden kuluttua leikkauksesta. Tutkimuksen perusteella lihavuusleikkaus parantaa tehokkaasti maksan aineenvaihduntaa. Vaikka rasvamaksa paranee leikkauksen jälkeen, pysyy maksan rasvahappojen soluunotto lisääntyneenä, mikä viittaa siihen, että rasvahapot käytetään energian tuotantoon sen sijaan että ne varastoitaisiin. Lisääntynyt porttilaskimon verenvirtaus saattaa olla yhteydessä parantuneeseen maksan aineenvaihduntaan leikkauksen jälkeen. Lisäksi tulokset viittaavat aivomaksa -akselin olemassaoloon. Sillä voi olla merkitystä elimistön glukoosiaineenvaihdunnan heikkenemisessä

Evidence for a role of the molecular chaperone clusterin in systemic amyloidosis

Thesis (Ph.D.)--Boston UniversityThe systemic amyloidoses are a diverse collection of multi-organ diseases that feature misfolding and aggregation of an amyloidogenic protein leading to the shared, common manifestation of extracellular tissue amyloid fibril deposition. As the pathologic mechanisms are complex, molecular, biochemical and biophysical studies of these disease processes are needed to identify factors other than the amyloidogenic protein that may be important. The investigations presented in this thesis have explored the involvement of clusterin (CLU), a plasma protein, in transthyretin (TTR) and immunoglobulin light chain (LC) forms of systemic amyloidosis. CLU, ubiquitous in biological fluids, is an extracellular chaperone that is related to heat shock proteins. We hypothesized that CLU is part of a proteostatic mechanism to remove TTR or LC proteins from circulation by complexing with these amyloidogenic proteins to prevent proteotoxic stresses. Further, we posited that certain aspects of CLU biology, genetics, and biochemistry could be exploited for diagnostic or therapeutic strategies in the systemic amyloidoses. Analyses of amyloid deposits in SSA (senile systemic amyloidosis), ATTR (familial TTR-related amyloidosis), and AL (primary LC amyloidosis) tissues was accomplished using light and electron microscopic techniques. Results of these studies indicated that CLU is a component of TTR and LC amyloid deposits. The potential of CLU as a biomarker for amyloidosis was also investigated; plasma circulating CLU levels in patient sera was measured for correlation with disease status and CLU genetic variation within our patient population. To investigate the underlying biophysical and biochemical associations of CLU with amyloid fibril proteins, we purified CLU from pooled human serum and tested the ability of the protein to bind, stabilize, and prevent TTR misfolding and amyloid fibril formation. Direct binding of CLU to TTR oligomers was demonstrated using surface plasmon resonance. As observed by circular dichroism spectroscopy, CLU was capable of stabilizing TTR secondary structure. By quantitative Congo red binding, we showed that CLU inhibited TTR amyloid fibril formation. Together, these results provide evidence that CLU is a common element of SSA, ATTR, and AL amyloidosis pathology, can function as a circulating molecular chaperone, and is part of a proteostatic mechanism in amyloid fibril formation

Short linear motif candidates in the cell entry system used by SARS-CoV-2 and their potential therapeutic implications

The first reported receptor for SARS-CoV-2 on host cells was the angiotensin-converting enzyme 2 (ACE2). However, the viral spike protein also has an RGD motif, suggesting that cell surface integrins may be co-receptors. We examined the sequences of ACE2 and integrins with the Eukaryotic Linear Motif (ELM) resource and identified candidate short linear motifs (SLiMs) in their short, unstructured, cytosolic tails with potential roles in endocytosis, membrane dynamics, autophagy, cytoskeleton, and cell signaling. These SLiM candidates are highly conserved in vertebrates and may interact with the μ2 subunit of the endocytosis-associated AP2 adaptor complex, as well as with various protein domains (namely, I-BAR, LC3, PDZ, PTB, and SH2) found in human signaling and regulatory proteins. Several motifs overlap in the tail sequences, suggesting that they may act as molecular switches, such as in response to tyrosine phosphorylation status. Candidate LC3-interacting region (LIR) motifs are present in the tails of integrin β3 and ACE2, suggesting that these proteins could directly recruit autophagy components. Our findings identify several molecular links and testable hypotheses that could uncover mechanisms of SARS-CoV-2 attachment, entry, and replication against which it may be possible to develop host-directed therapies that dampen viral infection and disease progression. Several of these SLiMs have now been validated to mediate the predicted peptide interactions.Fil: Mészáros, Bálint. European Molecular Biology Laboratory; AlemaniaFil: Sámano Sánchez, Hugo. European Molecular Biology Laboratory; AlemaniaFil: Alvarado Valverde, Jesús. European Molecular Biology Laboratory; Alemania. Ruprecht Karls Universitat Heidelberg; AlemaniaFil: Čalyševa, Jelena. European Molecular Biology Laboratory; Alemania. Ruprecht Karls Universitat Heidelberg; AlemaniaFil: Martinez Perez, Elizabeth. Fundación Instituto Leloir; Argentina. European Molecular Biology Laboratory; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alves, Renato. European Molecular Biology Laboratory; AlemaniaFil: Shields, Denis C.. Universidad de Dublin; IrlandaFil: Kumar, Manjeet. European Molecular Biology Laboratory; AlemaniaFil: Rippmann, Friedrich. Computational Chemistry & Biology; AlemaniaFil: Chemes, Lucia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Gibson, Toby James. European Molecular Biology Laboratory; Alemani