TY - JOUR A1 - Nanda, Indrajit A1 - Schröder, Sarah K. A1 - Steinlein, Claus A1 - Haaf, Thomas A1 - Buhl, Eva M. A1 - Grimm, Domink G. A1 - Weiskirchen, Ralf T1 - Rat hepatic stellate cell line CFSC-2G: genetic markers and short tandem repeat profile useful for cell line authentication JF - Cells N2 - 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). KW - liver KW - extracellular matrix KW - hepatic stellate cell KW - myofibroblast KW - fibrosis KW - stress fibers KW - spectral karyotyping KW - rhodamine–phalloidin stain KW - next-generation sequencing KW - STR profile Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-288067 SN - 2073-4409 VL - 11 IS - 18 ER - TY - JOUR A1 - Nanda, Indrajit A1 - Steinlein, Claus A1 - Haaf, Thomas A1 - Buhl, Eva M. A1 - Grimm, Domink G. A1 - Friedman, Scott L. A1 - Meurer, Steffen K. A1 - Schröder, Sarah K. A1 - Weiskirchen, Ralf T1 - Genetic characterization of rat hepatic stellate cell line HSC-T6 for in vitro cell line authentication JF - Cells N2 - 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). KW - liver KW - extracellular matrix KW - hepatic stellate cell KW - myofibroblast KW - fibrosis KW - in vitro model KW - SKY analysis KW - phalloidin stain KW - next generation sequencing KW - STR profile Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-275178 SN - 2073-4409 VL - 11 IS - 11 ER - TY - JOUR A1 - Nanda, Indrajit A1 - Schories, Susanne A1 - Simeonov, Ivan A1 - Adolfi, Mateus Contar A1 - Du, Kang A1 - Steinlein, Claus A1 - Alsheimer, Manfred A1 - Haaf, Thomas A1 - Schartl, Manfred T1 - Evolution of the degenerated Y-chromosome of the swamp guppy, Micropoecilia picta JF - Cells N2 - 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. KW - sex chromosomes KW - heterochromatin KW - Y chromosome degeneration KW - meiosis KW - synaptonemal complex KW - recombination KW - 5-methylcytosine KW - testosterone KW - sexual antagonistic genes KW - sex linked pigmentation pattern Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-267242 SN - 2073-4409 VL - 11 IS - 7 ER - TY - JOUR A1 - Zaum, Ann‐Kathrin A1 - Nanda, Indrajit A1 - Kress, Wolfram A1 - Rost, Simone T1 - Detection of pericentric inversion with breakpoint in DMD by whole genome sequencing JF - Molecular Genetics & Genomic Medicine N2 - 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. KW - chromosome inversion KW - Duchenne muscular dystrophy KW - dystrophin KW - genetic diagnostics KW - whole genome sequencing Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-293940 VL - 10 IS - 10 ER - TY - JOUR A1 - Fiedler, David A1 - Hirsch, Daniela A1 - El Hajj, Nady A1 - Yang, Howard H. A1 - Hu, Yue A1 - Sticht, Carsten A1 - Nanda, Indrajit A1 - Belle, Sebastian A1 - Rueschoff, Josef A1 - Lee, Maxwell P. A1 - Ried, Thomas A1 - Haaf, Thomas A1 - Gaiser, Timo T1 - Genome‐wide DNA methylation analysis of colorectal adenomas with and without recurrence reveals an association between cytosine‐phosphate‐guanine methylation and histological subtypes JF - Genes, Chromosomes and Cancer N2 - Aberrant methylation of DNA is supposed to be a major and early driver of colonic adenoma development, which may result in colorectal cancer (CRC). Although gene methylation assays are used already for CRC screening, differential epigenetic alterations of recurring and nonrecurring colorectal adenomas have yet not been systematically investigated. Here, we collected a sample set of formalin‐fixed paraffin‐embedded colorectal low‐grade adenomas (n = 72) consisting of primary adenomas without and with recurrence (n = 59), recurrent adenomas (n = 10), and normal mucosa specimens (n = 3). We aimed to unveil differentially methylated CpG positions (DMPs) across the methylome comparing not only primary adenomas without recurrence vs primary adenomas with recurrence but also primary adenomas vs recurrent adenomas using the Illumina Human Methylation 450K BeadChip array. Unsupervised hierarchical clustering exhibited a significant association of methylation patterns with histological adenoma subtypes. No significant DMPs were identified comparing primary adenomas with and without recurrence. Despite that, a total of 5094 DMPs (false discovery rate <0.05; fold change >10%) were identified in the comparisons of recurrent adenomas vs primary adenomas with recurrence (674; 98% hypermethylated), recurrent adenomas vs primary adenomas with and without recurrence (241; 99% hypermethylated) and colorectal adenomas vs normal mucosa (4179; 46% hypermethylated). DMPs in cytosine‐phosphate‐guanine (CpG) islands were frequently hypermethylated, whereas open sea‐ and shelf‐regions exhibited hypomethylation. Gene ontology analysis revealed enrichment of genes associated with the immune system, inflammatory processes, and cancer pathways. In conclusion, our methylation data could assist in establishing a more robust and reproducible histological adenoma classification, which is a prerequisite for improving surveillance guidelines. KW - adenoma KW - DNA methylation KW - epigenetics KW - histological subtype KW - recurrence Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-212676 VL - 58 IS - 11 SP - 783 EP - 797 ER - TY - JOUR A1 - El Hajj, Nady A1 - Dittrich, Marcus A1 - Böck, Julia A1 - Kraus, Theo F. J. A1 - Nanda, Indrajit A1 - Müller, Tobias A1 - Seidmann, Larissa A1 - Tralau, Tim A1 - Galetzka, Danuta A1 - Schneider, Eberhard A1 - Haaf, Thomas T1 - Epigenetic dysregulation in the developing Down syndrome cortex JF - Epigenetics N2 - Using Illumina 450K arrays, 1.85% of all analyzed CpG sites were significantly hypermethylated and 0.31% hypomethylated in fetal Down syndrome (DS) cortex throughout the genome. The methylation changes on chromosome 21 appeared to be balanced between hypo- and hyper-methylation, whereas, consistent with prior reports, all other chromosomes showed 3-11times more hyper- than hypo-methylated sites. Reduced NRSF/REST expression due to upregulation of DYRK1A (on chromosome 21q22.13) and methylation of REST binding sites during early developmental stages may contribute to this genome-wide excess of hypermethylated sites. Upregulation of DNMT3L (on chromosome 21q22.4) could lead to de novo methylation in neuroprogenitors, which then persists in the fetal DS brain where DNMT3A and DNMT3B become downregulated. The vast majority of differentially methylated promoters and genes was hypermethylated in DS and located outside chromosome 21, including the protocadherin gamma (PCDHG) cluster on chromosome 5q31, which is crucial for neural circuit formation in the developing brain. Bisulfite pyrosequencing and targeted RNA sequencing showed that several genes of PCDHG subfamilies A and B are hypermethylated and transcriptionally downregulated in fetal DS cortex. Decreased PCDHG expression is expected to reduce dendrite arborization and growth in cortical neurons. Since constitutive hypermethylation of PCDHG and other genes affects multiple tissues, including blood, it may provide useful biomarkers for DS brain development and pharmacologic targets for therapeutic interventions. KW - trisomy 21 KW - DNA methylation KW - Down syndrome KW - fetal brain development KW - frontal cortex KW - protocadherin gamma cluster Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-191239 VL - 11 IS - 8 ER - TY - JOUR A1 - Doll, Julia A1 - Vona, Barbara A1 - Schnapp, Linda A1 - Rüschendorf, Franz A1 - Khan, Imran A1 - Khan, Saadullah A1 - Muhammad, Noor A1 - Alam Khan, Sher A1 - Nawaz, Hamed A1 - Khan, Ajmal A1 - Ahmad, Naseer A1 - Kolb, Susanne M. A1 - Kühlewein, Laura A1 - Labonne, Jonathan D. J. A1 - Layman, Lawrence C. A1 - Hofrichter, Michaela A. H. A1 - Röder, Tabea A1 - Dittrich, Marcus A1 - Müller, Tobias A1 - Graves, Tyler D. A1 - Kong, Il-Keun A1 - Nanda, Indrajit A1 - Kim, Hyung-Goo A1 - Haaf, Thomas T1 - Genetic Spectrum of Syndromic and Non-Syndromic Hearing Loss in Pakistani Families JF - Genes N2 - The current molecular genetic diagnostic rates for hereditary hearing loss (HL) vary considerably according to the population background. Pakistan and other countries with high rates of consanguineous marriages have served as a unique resource for studying rare and novel forms of recessive HL. A combined exome sequencing, bioinformatics analysis, and gene mapping approach for 21 consanguineous Pakistani families revealed 13 pathogenic or likely pathogenic variants in the genes GJB2, MYO7A, FGF3, CDC14A, SLITRK6, CDH23, and MYO15A, with an overall resolve rate of 61.9%. GJB2 and MYO7A were the most frequently involved genes in this cohort. All the identified variants were either homozygous or compound heterozygous, with two of them not previously described in the literature (15.4%). Overall, seven missense variants (53.8%), three nonsense variants (23.1%), two frameshift variants (15.4%), and one splice-site variant (7.7%) were observed. Syndromic HL was identified in five (23.8%) of the 21 families studied. This study reflects the extreme genetic heterogeneity observed in HL and expands the spectrum of variants in deafness-associated genes. KW - genetic diagnosis KW - consanguinity KW - genome-wide linkage analysis KW - hearing loss KW - Pakistan KW - exome sequencing Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-219293 SN - 2073-4425 VL - 11 IS - 11 ER - TY - JOUR A1 - Schneider, Eberhard A1 - Dittrich, Marcus A1 - Böck, Julia A1 - Nanda, Indrajit A1 - Müller, Tobias A1 - Seidmann, Larissa A1 - Tralau, Tim A1 - Galetzka, Danuta A1 - El Hajj, Nady A1 - Haaf, Thomas T1 - CpG sites with continuously increasing or decreasing methylation from early to late human fetal brain development JF - Gene N2 - Normal human brain development is dependent on highly dynamic epigenetic processes for spatial and temporal gene regulation. Recent work identified wide-spread changes in DNA methylation during fetal brain development. We profiled CpG methylation in frontal cortex of 27 fetuses from gestational weeks 12-42, using Illumina 450K methylation arrays. Sites showing genome-wide significant correlation with gestational age were compared to a publicly available data set from gestational weeks 3-26. Altogether, we identified 2016 matching developmentally regulated differentially methylated positions (m-dDMPs): 1767 m-dDMPs were hypermethylated and 1149 hypomethylated during fetal development. M-dDMPs are underrepresented in CpG islands and gene promoters, and enriched in gene bodies. They appear to cluster in certain chromosome regions. M-dDMPs are significantly enriched in autism-associated genes and CpGs. Our results promote the idea that reduced methylation dynamics during fetal brain development may predispose to autism. In addition, m-dDMPs are enriched in genes with human-specific brain expression patterns and/or histone modifications. Collectively, we defined a subset of dDMPs exhibiting constant methylation changes from early to late pregnancy. The same epigenetic mechanisms involving methylation changes in cis-regulatory regions may have been adopted for human brain evolution and ontogeny. KW - Autism spectrum disorders KW - DNA methylation KW - Genome KW - Autism KW - Frontal cortex KW - Human prefrontal cortex KW - Gene-expression KW - Schizophrenia KW - Patterns KW - Transcription KW - Epigenetics KW - Environment KW - Fetal brain development KW - DNA methylation dynamics KW - Methylome Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-186936 VL - 592 IS - 1 ER - TY - JOUR A1 - Maierhofer, Anna A1 - Flunkert, Julia A1 - Oshima, Junko A1 - Martin, George M. A1 - Poot, Martin A1 - Nanda, Indrajit A1 - Dittrich, Marcus A1 - Müller, Tobias A1 - Haaf, Thomas T1 - Epigenetic signatures of Werner syndrome occur early in life and are distinct from normal epigenetic aging processes JF - Aging Cell N2 - Werner Syndrome (WS) is an adult‐onset segmental progeroid syndrome. Bisulfite pyrosequencing of repetitive DNA families revealed comparable blood DNA methylation levels between classical (18 WRN‐mutant) or atypical WS (3 LMNA‐mutant and 3 POLD1‐mutant) patients and age‐ and sex‐matched controls. WS was not associated with either age‐related accelerated global losses of ALU, LINE1, and α‐satellite DNA methylations or gains of rDNA methylation. Single CpG methylation was analyzed with Infinium MethylationEPIC arrays. In a correspondence analysis, atypical WS samples clustered together with the controls and were clearly separated from classical WS, consistent with distinct epigenetic pathologies. In classical WS, we identified 659 differentially methylated regions (DMRs) comprising 3,656 CpG sites and 613 RefSeq genes. The top DMR was located in the HOXA4 promoter. Additional DMR genes included LMNA, POLD1, and 132 genes which have been reported to be differentially expressed in WRN‐mutant/depleted cells. DMRs were enriched in genes with molecular functions linked to transcription factor activity and sequence‐specific DNA binding to promoters transcribed by RNA polymerase II. We propose that transcriptional misregulation of downstream genes by the absence of WRN protein contributes to the variable premature aging phenotypes of WS. There were no CpG sites showing significant differences in DNA methylation changes with age between WS patients and controls. Genes with both WS‐ and age‐related methylation changes exhibited a constant offset of methylation between WRN‐mutant patients and controls across the entire analyzed age range. WS‐specific epigenetic signatures occur early in life and do not simply reflect an acceleration of normal epigenetic aging processes. KW - (classical and atypical) Werner syndrome KW - bisulfite pyrosequencing KW - methylation array KW - premature aging KW - segmental progeria KW - transcription deficiency Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202733 VL - 18 ER - TY - JOUR A1 - Lekszas, Caroline A1 - Nanda, Indrajit A1 - Vona, Barbara A1 - Böck, Julia A1 - Ashrafzadeh, Farah A1 - Donyadideh, Nahid A1 - Ebrahimzadeh, Farnoosh A1 - Ahangari, Najmeh A1 - Maroofian, Reza A1 - Karimiani, Ehsan Ghayoor A1 - Haaf, Thomas T1 - Unbalanced segregation of a paternal t(9;11)(p24.3;p15.4) translocation causing familial Beckwith-Wiedemann syndrome: a case report JF - BMC Medical Genomics N2 - Background The vast majority of cases with Beckwith-Wiedemann syndrome (BWS) are caused by a molecular defect in the imprinted chromosome region 11p15.5. The underlying mechanisms include epimutations, uniparental disomy, copy number variations, and structural rearrangements. In addition, maternal loss-of-function mutations in CDKN1C are found. Despite growing knowledge on BWS pathogenesis, up to 20% of patients with BWS phenotype remain without molecular diagnosis. Case presentation Herein, we report an Iranian family with two females affected with BWS in different generations. Bisulfite pyrosequencing revealed hypermethylation of the H19/IGF2: intergenic differentially methylated region (IG DMR), also known as imprinting center 1 (IC1) and hypomethylation of the KCNQ1OT1: transcriptional start site (TSS) DMR (IC2). Array CGH demonstrated an 8 Mb duplication on chromosome 11p15.5p15.4 (205,827-8,150,933) and a 1 Mb deletion on chromosome 9p24.3 (209,020-1,288,114). Chromosome painting revealed that this duplication-deficiency in both patients is due to unbalanced segregation of a paternal reciprocal t(9;11)(p24.3;p15.4) translocation. Conclusions This is the first report of a paternally inherited unbalanced translocation between the chromosome 9 and 11 short arms underlying familial BWS. Copy number variations involving the 11p15.5 region are detected by the consensus diagnostic algorithm. However, in complex cases which do not only affect the BWS region itself, characterization of submicroscopic chromosome rearrangements can assist to estimate the recurrence risk and possible phenotypic outcomes. KW - Familial Beckwith-Wiedemann syndrome KW - copy number variation KW - duplication-deficiency KW - genomic imprinting KW - submicroscopic chromosome rearrangement KW - reciprocal translocation Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200422 VL - 12 ER -