@article{DollKolbSchnappetal.2020, author = {Doll, Julia and Kolb, Susanne and Schnapp, Linda and Rad, Aboulfazl and R{\"u}schendorf, Franz and Khan, Imran and Adli, Abolfazl and Hasanzadeh, Atefeh and Liedtke, Daniel and Knaup, Sabine and Hofrichter, Michaela AH and M{\"u}ller, Tobias and Dittrich, Marcus and Kong, Il-Keun and Kim, Hyung-Goo and Haaf, Thomas and Vona, Barbara}, title = {Novel loss-of-function variants in CDC14A are associated with recessive sensorineural hearing loss in Iranian and Pakistani patients}, series = {International Journal of Molecular Sciences}, volume = {21}, journal = {International Journal of Molecular Sciences}, number = {1}, issn = {1422-0067}, doi = {10.3390/ijms21010311}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-285142}, year = {2020}, abstract = {CDC14A encodes the Cell Division Cycle 14A protein and has been associated with autosomal recessive non-syndromic hearing loss (DFNB32), as well as hearing impairment and infertile male syndrome (HIIMS) since 2016. To date, only nine variants have been associated in patients whose initial symptoms included moderate-to-profound hearing impairment. Exome analysis of Iranian and Pakistani probands who both showed bilateral, sensorineural hearing loss revealed a novel splice site variant (c.1421+2T>C, p.?) that disrupts the splice donor site and a novel frameshift variant (c.1041dup, p.Ser348Glnfs*2) in the gene CDC14A, respectively. To evaluate the pathogenicity of both loss-of-function variants, we analyzed the effects of both variants on the RNA-level. The splice variant was characterized using a minigene assay. Altered expression levels due to the c.1041dup variant were assessed using RT-qPCR. In summary, cDNA analysis confirmed that the c.1421+2T>C variant activates a cryptic splice site, resulting in a truncated transcript (c.1414_1421del, p.Val472Leufs*20) and the c.1041dup variant results in a defective transcript that is likely degraded by nonsense-mediated mRNA decay. The present study functionally characterizes two variants and provides further confirmatory evidence that CDC14A is associated with a rare form of hereditary hearing loss.}, 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{MaierhoferFlunkertDittrichetal.2017, author = {Maierhofer, Anna and Flunkert, Julia and Dittrich, Marcus and M{\"u}ller, Tobias and Schindler, Detlev and Nanda, Indrajit and Haaf, Thomas}, title = {Analysis of global DNA methylation changes in primary human fibroblasts in the early phase following X-ray irradiation}, series = {PLoS ONE}, volume = {12}, journal = {PLoS ONE}, number = {5}, doi = {10.1371/journal.pone.0177442}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170895}, pages = {e0177442}, year = {2017}, abstract = {Epigenetic alterations may contribute to the generation of cancer cells in a multi-step process of tumorigenesis following irradiation of normal body cells. Primary human fibroblasts with intact cell cycle checkpoints were used as a model to test whether X-ray irradiation with 2 and 4 Gray induces direct epigenetic effects (within the first cell cycle) in the exposed cells. ELISA-based fluorometric assays were consistent with slightly reduced global DNA methylation and hydroxymethylation, however the observed between-group differences were usually not significant. Similarly, bisulfite pyrosequencing of interspersed LINE-1 repeats and centromeric α-satellite DNA did not detect significant methylation differences between irradiated and non-irradiated cultures. Methylation of interspersed ALU repeats appeared to be slightly increased (one percentage point; p = 0.01) at 6 h after irradiation with 4 Gy. Single-cell analysis showed comparable variations in repeat methylation among individual cells in both irradiated and control cultures. Radiation-induced changes in global repeat methylation, if any, were much smaller than methylation variation between different fibroblast strains. Interestingly, α-satellite DNA methylation positively correlated with gestational age. Finally, 450K methylation arrays mainly targeting genes and CpG islands were used for global DNA methylation analysis. There were no detectable methylation differences in genic (promoter, 5' UTR, first exon, gene body, 3' UTR) and intergenic regions between irradiated and control fibroblast cultures. Although we cannot exclude minor effects, i.e. on individual CpG sites, collectively our data suggest that global DNA methylation remains rather stable in irradiated normal body cells in the early phase of DNA damage response.}, language = {en} } @article{VonaMaroofianBellacchioetal.2018, author = {Vona, Barbara and Maroofian, Reza and Bellacchio, Emanuele and Najafi, Maryam and Thompson, Kyle and Alahmad, Ahmad and He, Langping and Ahangari, Najmeh and Rad, Abolfazl and Shahrokhzadeh, Sima and Bahena, Paulina and Mittag, Falk and Traub, Frank and Movaffagh, Jebrail and Amiri, Nafise and Doosti, Mohammad and Boostani, Reza and Shirzadeh, Ebrahim and Haaf, Thomas and Diodato, Daria and Schmidts, Miriam and Taylor, Robert W. and Karimiani, Ehsan Ghayoor}, title = {Expanding the clinical phenotype of IARS2-related mitochondrial disease}, series = {BMC Medical Genetics}, volume = {19}, journal = {BMC Medical Genetics}, number = {196}, doi = {10.1186/s12881-018-0709-3}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-176620}, year = {2018}, abstract = {Background: IARS2 encodes a mitochondrial isoleucyl-tRNA synthetase, a highly conserved nuclear-encoded enzyme required for the charging of tRNAs with their cognate amino acid for translation. Recently, pathogenic IARS2 variants have been identified in a number of patients presenting broad clinical phenotypes with autosomal recessive inheritance. These phenotypes range from Leigh and West syndrome to a new syndrome abbreviated CAGSSS that is characterised by cataracts, growth hormone deficiency, sensory neuropathy, sensorineural hearing loss, and skeletal dysplasia, as well as cataract with no additional anomalies. Methods: Genomic DNA from Iranian probands from two families with consanguineous parental background and overlapping CAGSSS features were subjected to exome sequencing and bioinformatics analysis. Results: Exome sequencing and data analysis revealed a novel homozygous missense variant (c.2625C > T, p.Pro909Ser, NM_018060.3) within a 14.3 Mb run of homozygosity in proband 1 and a novel homozygous missense variant (c.2282A > G, p.His761Arg) residing in an ~ 8 Mb region of homozygosity in a proband of the second family. Patient-derived fibroblasts from proband 1 showed normal respiratory chain enzyme activity, as well as unchanged oxidative phosphorylation protein subunits and IARS2 levels. Homology modelling of the known and novel amino acid residue substitutions in IARS2 provided insight into the possible consequence of these variants on function and structure of the protein. Conclusions: This study further expands the phenotypic spectrum of IARS2 pathogenic variants to include two patients (patients 2 and 3) with cataract and skeletal dysplasia and no other features of CAGSSS to the possible presentation of the defects in IARS2. Additionally, this study suggests that adult patients with CAGSSS may manifest central adrenal insufficiency and type II esophageal achalasia and proposes that a variable sensorineural hearing loss onset, proportionate short stature, polyneuropathy, and mild dysmorphic features are possible, as seen in patient 1. Our findings support that even though biallelic IARS2 pathogenic variants can result in a distinctive, clinically recognisable phenotype in humans, it can also show a wide range of clinical presentation from severe pediatric neurological disorders of Leigh and West syndrome to both non-syndromic cataract and cataract accompanied by skeletal dysplasia.}, language = {en} } @article{VonaNandaShehataDieleretal.2017, author = {Vona, Barbara and Nanda, Indrajit and Shehata-Dieler, Wafaa and Haaf, Thomas}, title = {Genetics of Tinnitus: Still in its Infancy}, series = {Frontiers in Neuroscience}, volume = {11}, journal = {Frontiers in Neuroscience}, number = {236}, doi = {10.3389/fnins.2017.00236}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170926}, year = {2017}, abstract = {Tinnitus is the perception of a phantom sound that affects between 10 and 15\% of the general population. Despite this considerable prevalence, treatments for tinnitus are presently lacking. Tinnitus exhibits a diverse array of recognized risk factors and extreme clinical heterogeneity. Furthermore, it can involve an unknown number of auditory and non-auditory networks and molecular pathways. This complex combination has hampered advancements in the field. The identification of specific genetic factors has been at the forefront of several research investigations in the past decade. Nine studies have examined genes in a case-control association approach. Recently, a genome-wide association study has highlighted several potentially significant pathways that are implicated in tinnitus. Two twin studies have calculated a moderate heritability for tinnitus and disclosed a greater concordance rate in monozygotic twins compared to dizygotic twins. Despite the more recent data alluding to genetic factors in tinnitus, a strong association with any specific genetic locus is lacking and a genetic study with sufficient statistical power has yet to be designed. Future research endeavors must overcome the many inherent limitations in previous study designs. This review summarizes the previously embarked upon tinnitus genetic investigations and summarizes the hurdles that have been encountered. The identification of candidate genes responsible for tinnitus may afford gene based diagnostic approaches, effective therapy development, and personalized therapeutic intervention.}, language = {en} } @article{PfeifferKruegerMaierhoferetal.2016, author = {Pfeiffer, Susanne and Kr{\"u}ger, Jacqueline and Maierhofer, Anna and B{\"o}ttcher, Yvonne and Kl{\"o}ting, Nora and El Hajj, Nady and Schleinitz, Dorit and Sch{\"o}n, Michael R. and Dietrich, Arne and Fasshauer, Mathias and Lohmann, Tobias and Dreßler, Miriam and Stumvoll, Michael and Haaf, Thomas and Bl{\"u}her, Matthias and Kovacs, Peter}, title = {Hypoxia-inducible factor 3A gene expression and methylation in adipose tissue is related to adipose tissue dysfunction}, series = {Scientific Reports}, volume = {6}, journal = {Scientific Reports}, number = {27969}, doi = {10.1038/srep27969}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-167662}, year = {2016}, abstract = {Recently, a genome-wide analysis identified DNA methylation of the HIF3A (hypoxia-inducible factor 3A) as strongest correlate of BMI. Here we tested the hypothesis that HIF3A mRNA expression and CpG-sites methylation in adipose tissue (AT) and genetic variants in HIF3A are related to parameters of AT distribution and function. In paired samples of subcutaneous AT (SAT) and visceral AT (VAT) from 603 individuals, we measured HIF3A mRNA expression and analyzed its correlation with obesity and related traits. In subgroups of individuals, we investigated the effects on HIF3A genetic variants on its AT expression (N = 603) and methylation of CpG-sites (N = 87). HIF3A expression was significantly higher in SAT compared to VAT and correlated with obesity and parameters of AT dysfunction (including CRP and leucocytes count). HIF3A methylation at cg22891070 was significantly higher in VAT compared to SAT and correlated with BMI, abdominal SAT and VAT area. Rs8102595 showed a nominal significant association with AT HIF3A methylation levels as well as with obesity and fat distribution. HIF3A expression and methylation in AT are fat depot specific, related to obesity and AT dysfunction. Our data support the hypothesis that HIF pathways may play an important role in the development of AT dysfunction in obesity.}, language = {en} } @article{VonaHofrichterSchroederetal.2018, author = {Vona, Barbara and Hofrichter, Michaela A. H. and Schr{\"o}der, J{\"o}rg and Shehata-Dieler, Wafaa and Nanda, Indrajit and Haaf, Thomas}, title = {Hereditary hearing loss SNP-microarray pilot study}, series = {BMC Research Notes}, volume = {11}, journal = {BMC Research Notes}, number = {391}, doi = {10.1186/s13104-018-3466-7}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-176239}, year = {2018}, abstract = {Objectives: Despite recent advancements in diagnostic tools, the genomic landscape of hereditary hearing loss remains largely uncharacterized. One strategy to understand genome-wide aberrations includes the analysis of copy number variation that can be mapped using SNP-microarray technology. A growing collection of literature has begun to uncover the importance of copy number variation in hereditary hearing loss. This pilot study underpins a larger effort that involves the stage-wise analysis of hearing loss patients, many of whom have advanced to high-throughput sequencing analysis. Data description: Our data originate from the Infinium HumanOmni1-Quad v1.0 SNP-microarrays (Illumina) that provide useful markers for genome-wide association studies and copy number variation analysis. This dataset comprises a cohort of 108 individuals (99 with hearing loss, 9 normal hearing family members) for the purpose of understanding the genetic contribution of copy number variations to hereditary hearing loss. These anonymized SNP-microarray data have been uploaded to the NCBI Gene Expression Omnibus and are intended to benefit other investigators interested in aggregating platform-matched array patient datasets or as part of a supporting reference tool for other laboratories to better understand recurring copy number variations in other genetic disorders.}, language = {en} } @article{PrelogHilligardtSchmidtetal.2016, author = {Prelog, Martina and Hilligardt, Deborah and Schmidt, Christian A. and Przybylski, Grzegorz K. and Leierer, Johannes and Almanzar, Giovanni and El Hajj, Nady and Lesch, Klaus-Peter and Arolt, Volker and Zwanzger, Peter and Haaf, Thomas and Domschke, Katharina}, title = {Hypermethylation of FOXP3 Promoter and Premature Aging of the Immune System in Female Patients with Panic Disorder?}, series = {PLoS ONE}, volume = {11}, journal = {PLoS ONE}, number = {6}, doi = {10.1371/journal.pone.0157930}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179684}, year = {2016}, abstract = {Immunological abnormalities associated with pathological conditions, such as higher infection rates, inflammatory diseases, cancer or cardiovascular events are common in patients with panic disorder. In the present study, T cell receptor excision circles (TRECs), Forkhead-Box-Protein P3 gene (FOXP3) methylation of regulatory T cells (Tregs) and relative telomere lengths (RTLs) were investigated in a total and subsamples of 131 patients with panic disorder as compared to 131 age- and sex-matched healthy controls in order to test for a potential dysfunction and premature aging of the immune system in anxiety disorders. Significantly lower TRECs (p = 0.004) as well as significant hypermethylation of the FOXP3 promoter region (p = 0.005) were observed in female (but not in male) patients with panic disorder as compared to healthy controls. No difference in relative telomere length was discerned between patients and controls, but significantly shorter telomeres in females, smokers and older persons within the patient group. The presently observed reduced TRECs in panic disorder patients and FOXP3 hypermethylation in female patients with panic disorder potentially reflect impaired thymus and immunosuppressive Treg function, which might partly account for the known increased morbidity and mortality of anxiety disorders conferred by e.g. cancer and cardiovascular disorders.}, language = {en} } @article{DollVonaSchnappetal.2020, author = {Doll, Julia and Vona, Barbara and Schnapp, Linda and R{\"u}schendorf, Franz and Khan, Imran and Khan, Saadullah and Muhammad, Noor and Alam Khan, Sher and Nawaz, Hamed and Khan, Ajmal and Ahmad, Naseer and Kolb, Susanne M. and K{\"u}hlewein, Laura and Labonne, Jonathan D. J. and Layman, Lawrence C. and Hofrichter, Michaela A. H. and R{\"o}der, Tabea and Dittrich, Marcus and M{\"u}ller, Tobias and Graves, Tyler D. and Kong, Il-Keun and Nanda, Indrajit and Kim, Hyung-Goo and Haaf, Thomas}, title = {Genetic Spectrum of Syndromic and Non-Syndromic Hearing Loss in Pakistani Families}, series = {Genes}, volume = {11}, journal = {Genes}, number = {11}, issn = {2073-4425}, doi = {10.3390/genes11111329}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-219293}, year = {2020}, abstract = {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.}, 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} }