@article{PlutaHoffjanZimmeretal.2022, author = {Pluta, Natalie and Hoffjan, Sabine and Zimmer, Frederic and K{\"o}hler, Cornelia and L{\"u}cke, Thomas and Mohr, Jennifer and Vorgerd, Matthias and Nguyen, Hoa Huu Phuc and Atlan, David and Wolf, Beat and Zaum, Ann-Kathrin and Rost, Simone}, title = {Homozygous inversion on chromosome 13 involving SGCG detected by short read whole genome sequencing in a patient suffering from limb-girdle muscular dystrophy}, series = {Genes}, volume = {13}, journal = {Genes}, number = {10}, issn = {2073-4425}, doi = {10.3390/genes13101752}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-288122}, year = {2022}, abstract = {New techniques in molecular genetic diagnostics now allow for accurate diagnosis in a large proportion of patients with muscular diseases. Nevertheless, many patients remain unsolved, although the clinical history and/or the muscle biopsy give a clear indication of the involved genes. In many cases, there is a strong suspicion that the cause must lie in unexplored gene areas, such as deep-intronic or other non-coding regions. In order to find these changes, next-generation sequencing (NGS) methods are constantly evolving, making it possible to sequence entire genomes to reveal these previously uninvestigated regions. Here, we present a young woman who was strongly suspected of having a so far genetically unsolved sarcoglycanopathy based on her clinical history and muscle biopsy. Using short read whole genome sequencing (WGS), a homozygous inversion on chromosome 13 involving SGCG and LINC00621 was detected. The breakpoint in intron 2 of SGCG led to the absence of γ-sarcoglycan, resulting in the manifestation of autosomal recessive limb-girdle muscular dystrophy 5 (LGMDR5) in the young woman.}, language = {en} } @article{NandaSchroederSteinleinetal.2022, author = {Nanda, Indrajit and Schr{\"o}der, Sarah K. and Steinlein, Claus and Haaf, Thomas and Buhl, Eva M. and Grimm, Domink G. and Weiskirchen, Ralf}, title = {Rat hepatic stellate cell line CFSC-2G: genetic markers and short tandem repeat profile useful for cell line authentication}, series = {Cells}, volume = {11}, journal = {Cells}, number = {18}, issn = {2073-4409}, doi = {10.3390/cells11182900}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-288067}, year = {2022}, abstract = {Hepatic stellate cells (HSCs) are also known as lipocytes, fat-storing cells, perisinusoidal cells, or Ito cells. These liver-specific mesenchymal cells represent about 5\% to 8\% of all liver cells, playing a key role in maintaining the microenvironment of the hepatic sinusoid. Upon chronic liver injury or in primary culture, these cells become activated and transdifferentiate into a contractile phenotype, i.e., the myofibroblast, capable of producing and secreting large quantities of extracellular matrix compounds. Based on their central role in the initiation and progression of chronic liver diseases, cultured HSCs are valuable in vitro tools to study molecular and cellular aspects of liver diseases. However, the isolation of these cells requires special equipment, trained personnel, and in some cases needs approval from respective authorities. To overcome these limitations, several immortalized HSC lines were established. One of these cell lines is CFSC, which was originally established from cirrhotic rat livers induced by carbon tetrachloride. First introduced in 1991, this cell line and derivatives thereof (i.e., CFSC-2G, CFSC-3H, CFSC-5H, and CFSC-8B) are now used in many laboratories as an established in vitro HSC model. We here describe molecular features that are suitable for cell authentication. Importantly, chromosome banding and multicolor spectral karyotyping (SKY) analysis demonstrate that the CFSC-2G genome has accumulated extensive chromosome rearrangements and most chromosomes exist in multiple copies producing a pseudo-triploid karyotype. Furthermore, our study documents a defined short tandem repeat (STR) profile including 31 species-specific markers, and a list of genes expressed in CFSC-2G established by bulk mRNA next-generation sequencing (NGS).}, language = {en} } @article{DumontWeberLassalleJolyBeauparlantetal.2022, author = {Dumont, Martine and Weber-Lassalle, Nana and Joly-Beauparlant, Charles and Ernst, Corinna and Droit, Arnaud and Feng, Bing-Jian and Dubois, St{\´e}phane and Collin-Deschesnes, Annie-Claude and Soucy, Penny and Vall{\´e}e, Maxime and Fournier, Fr{\´e}d{\´e}ric and Lema{\c{c}}on, Audrey and Adank, Muriel A. and Allen, Jamie and Altm{\"u}ller, Janine and Arnold, Norbert and Ausems, Margreet G. E. M. and Berutti, Riccardo and Bolla, Manjeet K. and Bull, Shelley and Carvalho, Sara and Cornelissen, Sten and Dufault, Michael R. and Dunning, Alison M. and Engel, Christoph and Gehrig, Andrea and Geurts-Giele, Willemina R. R. and Gieger, Christian and Green, Jessica and Hackmann, Karl and Helmy, Mohamed and Hentschel, Julia and Hogervorst, Frans B. L. and Hollestelle, Antoinette and Hooning, Maartje J. and Horv{\´a}th, Judit and Ikram, M. Arfan and Kaulfuß, Silke and Keeman, Renske and Kuang, Da and Luccarini, Craig and Maier, Wolfgang and Martens, John W. M. and Niederacher, Dieter and N{\"u}rnberg, Peter and Ott, Claus-Eric and Peters, Annette and Pharoah, Paul D. P. and Ramirez, Alfredo and Ramser, Juliane and Riedel-Heller, Steffi and Schmidt, Gunnar and Shah, Mitul and Scherer, Martin and St{\"a}bler, Antje and Strom, Tim M. and Sutter, Christian and Thiele, Holger and van Asperen, Christi J. and van der Kolk, Lizet and van der Luijt, Rob B. and Volk, Alexander E. and Wagner, Michael and Waisfisz, Quinten and Wang, Qin and Wang-Gohrke, Shan and Weber, Bernhard H. F. and Devilee, Peter and Tavtigian, Sean and Bader, Gary D. and Meindl, Alfons and Goldgar, David E. and Andrulis, Irene L. and Schmutzler, Rita K. and Easton, Douglas F. and Schmidt, Marjanka K. and Hahnen, Eric and Simard, Jacques}, title = {Uncovering the contribution of moderate-penetrance susceptibility genes to breast cancer by whole-exome sequencing and targeted enrichment sequencing of candidate genes in women of European ancestry}, series = {Cancers}, volume = {14}, journal = {Cancers}, number = {14}, issn = {2072-6694}, doi = {10.3390/cancers14143363}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-281768}, year = {2022}, abstract = {Rare variants in at least 10 genes, including BRCA1, BRCA2, PALB2, ATM, and CHEK2, are associated with increased risk of breast cancer; however, these variants, in combination with common variants identified through genome-wide association studies, explain only a fraction of the familial aggregation of the disease. To identify further susceptibility genes, we performed a two-stage whole-exome sequencing study. In the discovery stage, samples from 1528 breast cancer cases enriched for breast cancer susceptibility and 3733 geographically matched unaffected controls were sequenced. Using five different filtering and gene prioritization strategies, 198 genes were selected for further validation. These genes, and a panel of 32 known or suspected breast cancer susceptibility genes, were assessed in a validation set of 6211 cases and 6019 controls for their association with risk of breast cancer overall, and by estrogen receptor (ER) disease subtypes, using gene burden tests applied to loss-of-function and rare missense variants. Twenty genes showed nominal evidence of association (p-value < 0.05) with either overall or subtype-specific breast cancer. Our study had the statistical power to detect susceptibility genes with effect sizes similar to ATM, CHEK2, and PALB2, however, it was underpowered to identify genes in which susceptibility variants are rarer or confer smaller effect sizes. Larger sample sizes would be required in order to identify such genes.}, language = {en} } @article{RolfesBordeMoellenhoffetal.2022, author = {Rolfes, Muriel and Borde, Julika and M{\"o}llenhoff, Kathrin and Kayali, Mohamad and Ernst, Corinna and Gehrig, Andrea and Sutter, Christian and Ramser, Juliane and Niederacher, Dieter and Horv{\´a}th, Judit and Arnold, Norbert and Meindl, Alfons and Auber, Bernd and Rump, Andreas and Wang-Gohrke, Shan and Ritter, Julia and Hentschel, Julia and Thiele, Holger and Altm{\"u}ller, Janine and N{\"u}rnberg, Peter and Rhiem, Kerstin and Engel, Christoph and Wappenschmidt, Barbara and Schmutzler, Rita K. and Hahnen, Eric and Hauke, Jan}, title = {Prevalence of cancer predisposition germline variants in male breast cancer patients: results of the German Consortium for Hereditary Breast and Ovarian Cancer}, series = {Cancers}, volume = {14}, journal = {Cancers}, number = {13}, issn = {2072-6694}, doi = {10.3390/cancers14133292}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-281758}, year = {2022}, abstract = {Male breast cancer (mBC) is associated with a high prevalence of pathogenic variants (PVs) in the BRCA2 gene; however, data regarding other BC predisposition genes are limited. In this retrospective multicenter study, we investigated the prevalence of PVs in BRCA1/2 and 23 non-BRCA1/2 genes using a sample of 614 patients with mBC, recruited through the centers of the German Consortium for Hereditary Breast and Ovarian Cancer. A high proportion of patients with mBC carried PVs in BRCA2 (23.0\%, 142/614) and BRCA1 (4.6\%, 28/614). The prevalence of BRCA1/2 PVs was 11.0\% in patients with mBC without a family history of breast and/or ovarian cancer. Patients with BRCA1/2 PVs did not show an earlier disease onset than those without. The predominant clinical presentation of tumor phenotypes was estrogen receptor (ER)-positive, progesterone receptor (PR)-positive, and HER2-negative (77.7\%); further, 10.2\% of the tumors were triple-positive, and 1.2\% were triple-negative. No association was found between ER/PR/HER2 status and BRCA1/2 PV occurrence. Comparing the prevalence of protein-truncating variants (PTVs) between patients with mBC and control data (ExAC, n = 27,173) revealed significant associations of PTVs in both BRCA1 and BRCA2 with mBC (BRCA1: OR = 17.04, 95\% CI = 10.54-26.82, p < 10\(^{-5}\); BRCA2: OR = 77.71, 95\% CI = 58.71-102.33, p < 10\(^{-5}\)). A case-control investigation of 23 non-BRCA1/2 genes in 340 BRCA1/2-negative patients and ExAC controls revealed significant associations of PTVs in CHEK2, PALB2, and ATM with mBC (CHEK2: OR = 3.78, 95\% CI = 1.59-7.71, p = 0.002; PALB2: OR = 14.77, 95\% CI = 5.02-36.02, p < 10\(^{-5}\); ATM: OR = 3.36, 95\% CI = 0.89-8.96, p = 0.04). Overall, our findings support the benefit of multi-gene panel testing in patients with mBC irrespective of their family history, age at disease onset, and tumor phenotype.}, language = {en} } @article{NandaSteinleinHaafetal.2022, author = {Nanda, Indrajit and Steinlein, Claus and Haaf, Thomas and Buhl, Eva M. and Grimm, Domink G. and Friedman, Scott L. and Meurer, Steffen K. and Schr{\"o}der, Sarah K. and Weiskirchen, Ralf}, title = {Genetic characterization of rat hepatic stellate cell line HSC-T6 for in vitro cell line authentication}, series = {Cells}, volume = {11}, journal = {Cells}, number = {11}, issn = {2073-4409}, doi = {10.3390/cells11111783}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-275178}, year = {2022}, abstract = {Immortalized hepatic stellate cells (HSCs) established from mouse, rat, and humans are valuable in vitro models for the biomedical investigation of liver biology. These cell lines are homogenous, thereby providing consistent and reproducible results. They grow more robustly than primary HSCs and provide an unlimited supply of proteins or nucleic acids for biochemical studies. Moreover, they can overcome ethical concerns associated with the use of animal and human tissue and allow for fostering of the 3R principle of replacement, reduction, and refinement proposed in 1959 by William M. S. Russell and Rex L. Burch. Nevertheless, working with continuous cell lines also has some disadvantages. In particular, there are ample examples in which genetic drift and cell misidentification has led to invalid data. Therefore, many journals and granting agencies now recommend proper cell line authentication. We herein describe the genetic characterization of the rat HSC line HSC-T6, which was introduced as a new in vitro model for the study of retinoid metabolism. The consensus chromosome markers, outlined primarily through multicolor spectral karyotyping (SKY), demonstrate that apart from the large derivative chromosome 1 (RNO1), at least two additional chromosomes (RNO4 and RNO7) are found to be in three copies in all metaphases. Additionally, we have defined a short tandem repeat (STR) profile for HSC-T6, including 31 species-specific markers. The typical features of these cells have been further determined by electron microscopy, Western blotting, and Rhodamine-Phalloidin staining. Finally, we have analyzed the transcriptome of HSC-T6 cells by mRNA sequencing (mRNA-Seq) using next generation sequencing (NGS).}, language = {en} } @article{PrellSenPotabattulaetal.2022, author = {Prell, Andreas and Sen, Mustafa Orkun and Potabattula, Ramya and Bernhardt, Laura and Dittrich, Marcus and Hahn, Thomas and Schorsch, Martin and Zacchini, Federica and Ptak, Grazyna Ewa and Niemann, Heiner and Haaf, Thomas}, title = {Species-specific paternal age effects and sperm methylation levels of developmentally important genes}, series = {Cells}, volume = {11}, journal = {Cells}, number = {4}, issn = {2073-4409}, doi = {10.3390/cells11040731}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-262301}, year = {2022}, abstract = {A growing number of sperm methylome analyses have identified genomic loci that are susceptible to paternal age effects in a variety of mammalian species, including human, bovine, and mouse. However, there is little overlap between different data sets. Here, we studied whether or not paternal age effects on the sperm epigenome have been conserved in mammalian evolution and compared methylation patterns of orthologous regulatory regions (mainly gene promoters) containing both conserved and non-conserved CpG sites in 94 human, 36 bovine, and 94 mouse sperm samples, using bisulfite pyrosequencing. We discovered three (NFKB2, RASGEF1C, and RPL6) age-related differentially methylated regions (ageDMRs) in humans, four (CHD7, HDAC11, PAK1, and PTK2B) in bovines, and three (Def6, Nrxn2, and Tbx19) in mice. Remarkably, the identified sperm ageDMRs were all species-specific. Most ageDMRs were in genomic regions with medium methylation levels and large methylation variation. Orthologous regions in species not showing this age effect were either hypermethylated (>80\%) or hypomethylated (<20\%). In humans and mice, ageDMRs lost methylation, whereas bovine ageDMRs gained methylation with age. Our results are in line with the hypothesis that sperm ageDMRs are in regions under epigenomic evolution and may be part of an epigenetic mechanism(s) for lineage-specific environmental adaptations and provide a solid basis for studies on downstream effects in the genes analyzed here.}, language = {en} } @article{WagenhaeuserRickertSommeretal.2022, author = {Wagenh{\"a}user, Laura and Rickert, Vanessa and Sommer, Claudia and Wanner, Christoph and Nordbeck, Peter and Rost, Simone and {\"U}{\c{c}}eyler, Nurcan}, title = {X-chromosomal inactivation patterns in women with Fabry disease}, series = {Molecular Genetics \& Genomic Medicine}, volume = {10}, journal = {Molecular Genetics \& Genomic Medicine}, number = {9}, doi = {10.1002/mgg3.2029}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-312795}, year = {2022}, abstract = {Background Although Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the α-galactosidase A gene (GLA), women may develop severe symptoms. We investigated X-chromosomal inactivation patterns (XCI) as a potential determinant of symptom severity in FD women. Patients and Methods We included 95 women with mutations in GLA (n = 18 with variants of unknown pathogenicity) and 50 related men, and collected mouth epithelial cells, venous blood, and skin fibroblasts for XCI analysis using the methylation status of the androgen receptor gene. The mutated X-chromosome was identified by comparison of samples from relatives. Patients underwent genotype categorization and deep clinical phenotyping of symptom severity. Results 43/95 (45\%) women carried mutations categorized as classic. The XCI pattern was skewed (i.e., ≥75:25\% distribution) in 6/87 (7\%) mouth epithelial cell samples, 31/88 (35\%) blood samples, and 9/27 (33\%) skin fibroblast samples. Clinical phenotype, α-galactosidase A (GAL) activity, and lyso-Gb3 levels did not show intergroup differences when stratified for X-chromosomal skewing and activity status of the mutated X-chromosome. Conclusions X-inactivation patterns alone do not reliably reflect the clinical phenotype of women with FD when investigated in biomaterial not directly affected by FD. However, while XCI patterns may vary between tissues, blood frequently shows skewing of XCI patterns.}, language = {en} } @article{ZinkSeewaldRohrbachetal.2022, author = {Zink, Miriam and Seewald, Anne and Rohrbach, Mareike and Brodehl, Andreas and Liedtke, Daniel and Williams, Tatjana and Childs, Sarah J. and Gerull, Brenda}, title = {Altered expression of TMEM43 causes abnormal cardiac structure and function in zebrafish}, series = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, number = {17}, issn = {1422-0067}, doi = {10.3390/ijms23179530}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-286025}, year = {2022}, abstract = {Arrhythmogenic cardiomyopathy (ACM) is an inherited heart muscle disease caused by heterozygous missense mutations within the gene encoding for the nuclear envelope protein transmembrane protein 43 (TMEM43). The disease is characterized by myocyte loss and fibro-fatty replacement, leading to life-threatening ventricular arrhythmias and sudden cardiac death. However, the role of TMEM43 in the pathogenesis of ACM remains poorly understood. In this study, we generated cardiomyocyte-restricted transgenic zebrafish lines that overexpress eGFP-linked full-length human wild-type (WT) TMEM43 and two genetic variants (c.1073C>T, p.S358L; c.332C>T, p.P111L) using the Tol2-system. Overexpression of WT and p.P111L-mutant TMEM43 was associated with transcriptional activation of the mTOR pathway and ribosome biogenesis, and resulted in enlarged hearts with cardiomyocyte hypertrophy. Intriguingly, mutant p.S358L TMEM43 was found to be unstable and partially redistributed into the cytoplasm in embryonic and adult hearts. Moreover, both TMEM43 variants displayed cardiac morphological defects at juvenile stages and ultrastructural changes within the myocardium, accompanied by dysregulated gene expression profiles in adulthood. Finally, CRISPR/Cas9 mutants demonstrated an age-dependent cardiac phenotype characterized by heart enlargement in adulthood. In conclusion, our findings suggest ultrastructural remodeling and transcriptomic alterations underlying the development of structural and functional cardiac defects in TMEM43-associated cardiomyopathy.}, language = {en} } @article{NandaSchoriesSimeonovetal.2022, author = {Nanda, Indrajit and Schories, Susanne and Simeonov, Ivan and Adolfi, Mateus Contar and Du, Kang and Steinlein, Claus and Alsheimer, Manfred and Haaf, Thomas and Schartl, Manfred}, title = {Evolution of the degenerated Y-chromosome of the swamp guppy, Micropoecilia picta}, series = {Cells}, volume = {11}, journal = {Cells}, number = {7}, issn = {2073-4409}, doi = {10.3390/cells11071118}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-267242}, year = {2022}, abstract = {The conspicuous colour sexual dimorphism of guppies has made them paradigmatic study objects for sex-linked traits and sex chromosome evolution. Both the X- and Y-chromosomes of the common guppy (Poecilia reticulata) are genetically active and homomorphic, with a large homologous part and a small sex specific region. This feature is considered to emulate the initial stage of sex chromosome evolution. A similar situation has been documented in the related Endler's and Oropuche guppies (P. wingei, P. obscura) indicating a common origin of the Y in this group. A recent molecular study in the swamp guppy (Micropoecilia. picta) reported a low SNP density on the Y, indicating Y-chromosome deterioration. We performed a series of cytological studies on M. picta to show that the Y-chromosome is quite small compared to the X and has accumulated a high content of heterochromatin. Furthermore, the Y-chromosome stands out in displaying CpG clusters around the centromeric region. These cytological findings evidently illustrate that the Y-chromosome in M. picta is indeed highly degenerated. Immunostaining for SYCP3 and MLH1 in pachytene meiocytes revealed that a substantial part of the Y remains associated with the X. A specific MLH1 hotspot site was persistently marked at the distal end of the associated XY structure. These results unveil a landmark of a recombining pseudoautosomal region on the otherwise strongly degenerated Y chromosome of M. picta. Hormone treatments of females revealed that, unexpectedly, no sexually antagonistic color gene is Y-linked in M. picta. All these differences to the Poecilia group of guppies indicate that the trajectories associated with the evolution of sex chromosomes are not in parallel.}, language = {en} } @article{LorenzMusacchioKunstmannetal.2022, author = {Lorenz, Delia and Musacchio, Thomas and Kunstmann, Erdmute and Grauer, Eva and Pluta, Natalie and Stock, Annika and Speer, Christian P. and Hebestreit, Helge}, title = {A case report of Sanfilippo syndrome - the long way to diagnosis}, series = {BMC Neurology}, volume = {22}, journal = {BMC Neurology}, number = {1}, doi = {10.1186/s12883-022-02611-7}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-300465}, year = {2022}, abstract = {Background Mucopolysaccharidosis type III (Sanfilippo syndrome) is a lysosomal storage disorder, caused by a deficiency in the heparan-N-sulfatase enzyme involved in the catabolism of the glycosaminoglycan heparan sulfate. It is characterized by early nonspecific neuropsychiatric symptoms, followed by progressive neurocognitive impairment in combination with only mild somatic features. In this patient group with a broad clinical spectrum a significant genotype-phenotype correlation with some mutations leading to a slower progressive, attenuated course has been demonstrated. Case presentation Our patient had complications in the neonatal period and was diagnosed with Mucopolysaccharidosis IIIa only at the age of 28 years. He was compound heterozygous for the variants p.R245H and p.S298P, the latter having been shown to lead to a significantly milder phenotype. Conclusions The diagnostic delay is even more prolonged in this patient population with comorbidities and a slowly progressive course of the disease.}, language = {en} } @article{ZaumNandaKressetal.2022, author = {Zaum, Ann-Kathrin and Nanda, Indrajit and Kress, Wolfram and Rost, Simone}, title = {Detection of pericentric inversion with breakpoint in DMD by whole genome sequencing}, series = {Molecular Genetics \& Genomic Medicine}, volume = {10}, journal = {Molecular Genetics \& Genomic Medicine}, number = {10}, doi = {10.1002/mgg3.2028}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-293940}, year = {2022}, abstract = {Background Dystrophinopathies caused by variants in the DMD gene are a well-studied muscle disease. The most common type of variant in DMD are large deletions. Very rarely reported forms of variants are chromosomal translocations, inversions and deep intronic variants (DIVs) because they are not detectable by standard diagnostic techniques (sequencing of coding sequence, copy number variant detection). This might be the reason that some clinically and histologically proven dystrophinopathy cases remain unsolved. Methods We used whole genome sequencing (WGS) to screen the entire DMD gene for variants in one of two brothers suffering from typical muscular dystrophy with strongly elevated creatine kinase levels. Results Although a pathogenic DIV could not be detected, we were able to identify a pericentric inversion with breakpoints in DMD intron 44 and Xq13.3, which could be confirmed by Sanger sequencing in the index as well as in his brother and mother. As this variation affects a major part of DMD it is most likely disease causing. Conclusion Our findings elucidate that WGS is capable of detecting large structural rearrangements and might be suitable for the genetic diagnostics of dystrophinopathies in the future. In particular, inversions might be a more frequent cause for dystrophinopathies as anticipated and should be considered in genetically unsolved dystrophinopathy cases.}, language = {en} } @phdthesis{Riekert2022, author = {Riekert, Elisa}, title = {Der Einfluss von Tnap auf die Zahnentwicklung im Zebrafisch (Danio rerio)}, doi = {10.25972/OPUS-28740}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-287406}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {Aufgrund mangelnder Aktivit{\"a}t der Gewebe-unspezifischen Phosphatase (tissue-nonspecific alkaline phosphatase, TNAP) kommt es zum Krankheitsbild der Hypophosphatasie (HPP). Neben skelettalen und neuronalen Symptomen leiden Patienten mit HPP h{\"a}ufig an einem vorzeitigen Verlust der Milchz{\"a}hne und weiteren dentalen Manifestationen, wie Zahnhartsubstanzdefekten, Eruptionsst{\"o}rungen, erweiterte Pulpenkammern oder einer verringerten alveol{\"a}ren Knochenh{\"o}he. Ziel der Arbeit war es, den Einfluss der TNAP auf die Zahnentwicklung von Zebrafischlarven zu untersuchen, um ein neues in-vivo Modell f{\"u}r die dentalen Auswirkungen bei Hypophosphatasie etablieren zu k{\"o}nnen. Um die sehr kleinen Z{\"a}hne der Zebrafischlarven auch in fr{\"u}hen Entwicklungsstadien darzustellen, wurden mittels verschiedener histologischer F{\"a}rbungen die Zahnstrukturen angef{\"a}rbt und die Larven danach in JB4®, einen polymeren Kunststoff, eingebettet. Im Anschluss wurden histologische Schnitte angefertigt und am Fluoreszenzmikroskop ausgewertet. Einerseits konnte durch In-situ-Hybridisierung die Expression verschiedener Gene, wie z.B. alpl (welches f{\"u}r die Tnap im Zebrafisch kodiert), im Bereich von dentalen Strukturen in verschiedenen Entwicklungsstadien nachgewiesen werden. Außerdem zeigte die Analyse der dentalen Strukturen nach Inhibition der Tnap mittels Levamisol bei f{\"u}nf Tage alten Zebrafischlarven eine Ver{\"a}nderung von Form, Gr{\"o}ße und Struktur der ersten Z{\"a}hne. Die TNAP-Inhibition f{\"u}hrte auch zur quantitativ nachweisbaren Steigerung des Fluoreszenzsignals von ß-Catenin, welches eine zentrale Funktion im Wnt/ß-Catenin-Signalweg besitzt und essenziell in verschiedenen zellul{\"a}ren Prozessen w{\"a}hrend der Embryogenese ist. Zusammenfassend zeigen die Ergebnisse der Arbeit, dass der Zebrafisch großes Potenzial als in-vivo Modell f{\"u}r die dentalen Symptome bei HPP bietet. Außerdem er{\"o}ffnen sich neue interessante Fragen in Bezug auf den Einfluss von ß-Catenin bei den fr{\"u}hen pathophysiologischen Prozessen der Erkrankung.}, subject = {Zebrab{\"a}rbling}, language = {de} } @phdthesis{Kiene2022, author = {Kiene, Carmen}, title = {Immunozytogenetische Analysen an Interphase-Zellen und Meiose-Stadien der Maus}, doi = {10.25972/OPUS-27910}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-279109}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {In der vorliegenden Arbeit wurden mittels 5-Methylcytosin Immunof{\"a}rbung zytogenetische Analysen an Metaphasechromosomen aus der Mitose, an Interphase-Zellen verschiedener Organe und an Meiose-Stadien der Maus (Mus musculus) zur Detektion hypermethylierter DNA durchgef{\"u}hrt. Zus{\"a}tzlich erfolgte eine C-B{\"a}nderung an Metaphasechromosomen und Meiose-Stadien zum Nachweis von konstitutivem Heterochromatin.}, subject = {Cytogenetik}, language = {de} } @article{BahenaDaftarianMaroofianetal.2022, author = {Bahena, Paulina and Daftarian, Narsis and Maroofian, Reza and Linares, Paola and Villalobos, Daniel and Mirrahimi, Mehraban and Rad, Aboulfazl and Doll, Julia and Hofrichter, Michaela A. H. and Koparir, Asuman and R{\"o}der, Tabea and Han, Seungbin and Sabbaghi, Hamideh and Ahmadieh, Hamid and Behboudi, Hassan and Villanueva-Mendoza, Cristina and Cort{\´e}s-Gonzalez, Vianney and Zamora-Ortiz, Rocio and Kohl, Susanne and Kuehlewein, Laura and Darvish, Hossein and Alehabib, Elham and La Arenas-Sordo, Maria de Luz and Suri, Fatemeh and Vona, Barbara and Haaf, Thomas}, title = {Unraveling the genetic complexities of combined retinal dystrophy and hearing impairment}, series = {Human Genetics}, volume = {141}, journal = {Human Genetics}, number = {3-4}, issn = {1432-1203}, doi = {10.1007/s00439-021-02303-1}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-267750}, pages = {785-803}, year = {2022}, abstract = {Usher syndrome, the most prevalent cause of combined hereditary vision and hearing impairment, is clinically and genetically heterogeneous. Moreover, several conditions with phenotypes overlapping Usher syndrome have been described. This makes the molecular diagnosis of hereditary deaf-blindness challenging. Here, we performed exome sequencing and analysis on 7 Mexican and 52 Iranian probands with combined retinal degeneration and hearing impairment (without intellectual disability). Clinical assessment involved ophthalmological examination and hearing loss questionnaire. Usher syndrome, most frequently due to biallelic variants in MYO7A (USH1B in 16 probands), USH2A (17 probands), and ADGRV1 (USH2C in 7 probands), was diagnosed in 44 of 59 (75\%) unrelated probands. Almost half of the identified variants were novel. Nine of 59 (15\%) probands displayed other genetic entities with dual sensory impairment, including Alstr{\"o}m syndrome (3 patients), cone-rod dystrophy and hearing loss 1 (2 probands), and Heimler syndrome (1 patient). Unexpected findings included one proband each with Scheie syndrome, coenzyme Q10 deficiency, and pseudoxanthoma elasticum. In four probands, including three Usher cases, dual sensory impairment was either modified/aggravated or caused by variants in distinct genes associated with retinal degeneration and/or hearing loss. The overall diagnostic yield of whole exome analysis in our deaf-blind cohort was 92\%. Two (3\%) probands were partially solved and only 3 (5\%) remained without any molecular diagnosis. In many cases, the molecular diagnosis is important to guide genetic counseling, to support prognostic outcomes and decisions with currently available and evolving treatment modalities.}, language = {en} } @phdthesis{Kuehl2022, author = {K{\"u}hl, Julia}, title = {FAAP100, der FA/BRCA-Signalweg f{\"u}r genomische Stabilit{\"a}t und das DNA-Reparatur-Netzwerk}, doi = {10.25972/OPUS-17166}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-171669}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {Die Fanconi-An{\"a}mie (FA) ist eine seltene, heterogene Erbkrankheit. Sie weist ein sehr variables klinisches Erscheinungsbild auf, das sich aus angeborenen Fehlbildungen, h{\"a}matologischen Funktionsst{\"o}rungen, einem erh{\"o}hten Risiko f{\"u}r Tumorentwicklung und endokrinen Pathologien zusammensetzt. Die Erkrankung z{\"a}hlt zu den genomischen Instabilit{\"a}tssyndromen, welche durch eine fehlerhafte DNA-Schadensreparatur gekennzeichnet sind. Bei der FA zeigt sich dies vor allem in einer charakteristischen Hypersensitivit{\"a}t gegen{\"u}ber DNA-quervernetzenden Substanzen (z. B. Mitomycin C, Cisplatin). Der zellul{\"a}re FA-Ph{\"a}notyp zeichnet sich durch eine erh{\"o}hte Chromosomenbr{\"u}chigkeit und einen Zellzyklusarrest in der G2-Phase aus. Diese Charakteristika sind bereits spontan vorhanden und werden durch Induktion mit DNA-quervernetzenden Substanzen verst{\"a}rkt. Der Gendefekt ist dabei in einem der 22 bekannten FA-Gene (FANCA, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L, -M, -N, -O, -P, -Q, -R, -S, -T, -U, -V, -W) oder in noch unbekannten FA-Genen zu finden. Die FA-Gendefekte werden mit Ausnahme von FANCR (dominant-negative de novo Mutationen) und FANCB (X-chromosomal) autosomal rezessiv vererbt. Die FA-Genprodukte bilden zusammen mit weiteren Proteinen den FA/BRCA-Signalweg. Das Schl{\"u}sselereignis dieses Signalwegs stellt die Monoubiquitinierung von FANCD2 und FANCI (ID2-Komplex) dar. Ausgehend davon l{\"a}sst sich zwischen upstream- und downstream-gelegenen FA-Proteinen unterscheiden. Letztere sind direkt an der DNA-Schadensreparatur beteiligt. Zu den upstream-gelegenen Proteinen z{\"a}hlt der FA-Kernkomplex, der sich aus bekannten FA-Proteinen und aus FA-assoziierten-Proteinen (FAAPs) zusammensetzt und f{\"u}r die Monoubiquitinierung des ID2-Komplexes verantwortlich ist. F{\"u}r FAAPs wurden bisher keine pathogenen humanen Mutationen beschrieben. Zu diesen Proteinen geh{\"o}rt auch FAAP100, das mit FANCB und FANCL innerhalb des FA-Kernkomplexes den Subkomplex LBP100 bildet. Durch die vorliegende Arbeit wurde eine n{\"a}here Charakterisierung dieses Proteins erreicht. In einer Amnion-Zelllinie konnte eine homozygote Missense-Mutation identifiziert werden. Der Fetus zeigte einen typischen FA-Ph{\"a}notyp und auch seine Zellen wiesen charakteristische FA-Merkmale auf. Der zellul{\"a}re Ph{\"a}notyp ließ sich durch FAAP100WT komplementieren, sodass die Pathogenit{\"a}t der Mutation bewiesen war. Unterst{\"u}tzend dazu wurden mithilfe des CRISPR/Cas9-Systems weitere FAAP100-defiziente Zelllinien generiert. Diese zeigten ebenfalls einen typischen FA-Ph{\"a}notyp, welcher sich durch FAAP100WT komplementieren ließ. Die in vitro-Modelle dienten als Grundlage daf{\"u}r, die Funktion des FA-Kernkomplexes im Allgemeinen und die des Subkomplexes LBP100 im Besonderen besser zu verstehen. Dabei kann nur durch intaktes FAAP100 das LBP100-Modul gebildet und dieses an die DNA-Schadensstelle transportiert werden. Dort leistet FAAP100 einen essentiellen Beitrag f{\"u}r den FANCD2-Monoubiquitinierungsprozess und somit f{\"u}r die Aktivierung der FA-abh{\"a}ngigen DNA-Schadensreparatur. Um die Funktion von FAAP100 auch in vivo zu untersuchen, wurde ein Faap100-/--Mausmodell generiert, das einen mit anderen FA-Mausmodellen vergleichbaren, relativ schweren FA-Ph{\"a}notyp aufwies. Aufgrund der Ergebnisse l{\"a}sst sich FAAP100 als neues FA-Gen klassifizieren. Zudem wurde die Rolle des Subkomplexes LBP100 innerhalb des FA-Kernkomplexes weiter aufgekl{\"a}rt. Beides tr{\"a}gt zu einem besseren Verst{\"a}ndnis des FA/BRCA-Signalweges bei. Ein weiterer Teil der vorliegenden Arbeit besch{\"a}ftigt sich mit der Charakterisierung von FAAP100138, einer bisher nicht validierten Isoform von FAAP100. Durch dieses Protein konnte der zellul{\"a}re FA-Ph{\"a}notyp von FAAP100-defizienten Zelllinien nicht komplementiert werden, jedoch wurden Hinweise auf einen dominant-negativen Effekt von FAAP100138 auf den FA/BRCA-Signalweg gefunden. Dies k{\"o}nnte zu der Erkl{\"a}rung beitragen, warum und wie der Signalweg, beispielsweise in bestimmtem Gewebearten, herunterreguliert wird. Zudem w{\"a}re eine Verwendung in der Krebstherapie denkbar.}, subject = {Fanconi-An{\"a}mie}, language = {de} }