Publications by authors named "Thomas Haaf"

158 Publications

Epigenetic age prediction in semen - marker selection and model development.

Aging (Albany NY) 2021 08 10;13(15):19145-19164. Epub 2021 Aug 10.

Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland.

DNA methylation analysis is becoming increasingly useful in biomedical research and forensic practice. The discovery of differentially methylated sites (DMSs) that continuously change over an individual's lifetime has led to breakthroughs in molecular age estimation. Although semen samples are often used in forensic DNA analysis, previous epigenetic age prediction studies mainly focused on somatic cell types. Here, Infinium MethylationEPIC BeadChip arrays were applied to semen-derived DNA samples, which identified numerous novel DMSs moderately correlated with age. Validation of the ten most age-correlated novel DMSs and three previously known sites in an independent set of semen-derived DNA samples using targeted bisulfite massively parallel sequencing, confirmed age-correlation for nine new and three previously known markers. Prediction modelling revealed the best model for semen, based on 6 CpGs from newly identified genes , , , and as well as the previously known gene, which predict age with a mean absolute error of 5.1 years in an independent test set. Further increases in the accuracy of age prediction from semen DNA will require technological progress to allow sensitive, simultaneous analysis of a much larger number of age correlated DMSs from the compromised DNA typical of forensic semen stains.
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http://dx.doi.org/10.18632/aging.203399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8386575PMC
August 2021

A piggyBac-based platform for genome editing and clonal rhesus macaque iPSC line derivation.

Sci Rep 2021 07 29;11(1):15439. Epub 2021 Jul 29.

Research Platform Degenerative Diseases, German Primate Center-Leibniz Institute for Primate Research, Kellnerweg 4, 37077, Göttingen, Germany.

Non-human primates (NHPs) are, due to their close phylogenetic relationship to humans, excellent animal models to study clinically relevant mutations. However, the toolbox for the genetic modification of NHPs is less developed than those for other species like mice. Therefore, it is necessary to further develop and refine genome editing approaches in NHPs. NHP pluripotent stem cells (PSCs) share key molecular signatures with the early embryo, which is an important target for genomic modification. Therefore, PSCs are a valuable test system for the validation of embryonic genome editing approaches. In the present study, we made use of the versatility of the piggyBac transposon system for different purposes in the context of NHP stem cell technology and genome editing. These include (1) Robust reprogramming of rhesus macaque fibroblasts to induced pluripotent stem cells (iPSCs); (2) Culture of the iPSCs under feeder-free conditions even after removal of the transgene resulting in transgene-free iPSCs; (3) Development of a CRISPR/Cas-based work-flow to edit the genome of rhesus macaque PSCs with high efficiency; (4) Establishment of a novel protocol for the derivation of gene-edited monoclonal NHP-iPSC lines. These findings facilitate efficient testing of genome editing approaches in NHP-PSC before their in vivo application.
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http://dx.doi.org/10.1038/s41598-021-94419-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8322147PMC
July 2021

Biallelic variants in KARS1 are associated with neurodevelopmental disorders and hearing loss recapitulated by the knockout zebrafish.

Genet Med 2021 Oct 25;23(10):1933-1943. Epub 2021 Jun 25.

Department of Pediatric Diseases, Mashhad University of Medical Sciences, Mashhad, Iran.

Purpose: Pathogenic variants in Lysyl-tRNA synthetase 1 (KARS1) have increasingly been recognized as a cause of early-onset complex neurological phenotypes. To advance the timely diagnosis of KARS1-related disorders, we sought to delineate its phenotype and generate a disease model to understand its function in vivo.

Methods: Through international collaboration, we identified 22 affected individuals from 16 unrelated families harboring biallelic likely pathogenic or pathogenic in KARS1 variants. Sequencing approaches ranged from disease-specific panels to genome sequencing. We generated loss-of-function alleles in zebrafish.

Results: We identify ten new and four known biallelic missense variants in KARS1 presenting with a moderate-to-severe developmental delay, progressive neurological and neurosensory abnormalities, and variable white matter involvement. We describe novel KARS1-associated signs such as autism, hyperactive behavior, pontine hypoplasia, and cerebellar atrophy with prevalent vermian involvement. Loss of kars1 leads to upregulation of p53, tissue-specific apoptosis, and downregulation of neurodevelopmental related genes, recapitulating key tissue-specific disease phenotypes of patients. Inhibition of p53 rescued several defects of kars1 knockouts.

Conclusion: Our work delineates the clinical spectrum associated with KARS1 defects and provides a novel animal model for KARS1-related human diseases revealing p53 signaling components as potential therapeutic targets.
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http://dx.doi.org/10.1038/s41436-021-01239-1DOI Listing
October 2021

Unraveling the genetic complexities of combined retinal dystrophy and hearing impairment.

Hum Genet 2021 Jun 20. Epub 2021 Jun 20.

Institute of Human Genetics, Julius Maximilians University Würzburg, 97074, Würzburg, Germany.

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 Alström 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.
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http://dx.doi.org/10.1007/s00439-021-02303-1DOI Listing
June 2021

Cereblon Enhancer Methylation and IMiD Resistance in Multiple Myeloma.

Blood 2021 Jun 11. Epub 2021 Jun 11.

University Hospital Würzburg, Würzburg, Germany.

Cereblon is the direct binding target of the immunomodulatory drugs that are commonly used to treat Multiple Myeloma, the second most frequent hematologic malignancy. Patients respond well to initial IMiD treatment but virtually all develop drug resistance over time with the underlying mechanisms poorly understood. We identified a yet undescribed DNA hypermethylation in an active intronic CRBN enhancer. Differential hypermethylation in this region was found increased in healthy plasma cells, but more pronounced in IMiD refractory MM. Methylation significantly correlated with decreased CRBN expression levels. DNTMi in vitro experiments induced CRBN enhancer demethylation and sensitizing effects on Lenalidomide treatment were observed in two MM cell lines. Thus, we provide first evidence that aberrant CRBN DNA methylation is a novel mechanism of IMiD resistance in Multiple Myeloma and may predict IMiD response prior treatment.
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http://dx.doi.org/10.1182/blood.2020010452DOI Listing
June 2021

Bi-allelic variants in IPO8 cause a connective tissue disorder associated with cardiovascular defects, skeletal abnormalities, and immune dysregulation.

Am J Hum Genet 2021 06 18;108(6):1126-1137. Epub 2021 May 18.

Victorian Clinical Genetics Services, Murdoch Children's Research Institute, Parkville 3052, Melbourne, VIC, Australia; Department of Paediatrics, The University of Melbourne, 3010 Parkville, Melbourne, VIC, Australia.

Dysregulated transforming growth factor TGF-β signaling underlies the pathogenesis of genetic disorders affecting the connective tissue such as Loeys-Dietz syndrome. Here, we report 12 individuals with bi-allelic loss-of-function variants in IPO8 who presented with a syndromic association characterized by cardio-vascular anomalies, joint hyperlaxity, and various degree of dysmorphic features and developmental delay as well as immune dysregulation; the individuals were from nine unrelated families. Importin 8 belongs to the karyopherin family of nuclear transport receptors and was previously shown to mediate TGF-β-dependent SMADs trafficking to the nucleus in vitro. The important in vivo role of IPO8 in pSMAD nuclear translocation was demonstrated by CRISPR/Cas9-mediated inactivation in zebrafish. Consistent with IPO8's role in BMP/TGF-β signaling, ipo8 zebrafish presented mild to severe dorso-ventral patterning defects during early embryonic development. Moreover, ipo8 zebrafish displayed severe cardiovascular and skeletal defects that mirrored the human phenotype. Our work thus provides evidence that IPO8 plays a critical and non-redundant role in TGF-β signaling during development and reinforces the existing link between TGF-β signaling and connective tissue defects.
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http://dx.doi.org/10.1016/j.ajhg.2021.04.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8206386PMC
June 2021

Lipid droplets in mammalian eggs are utilized during embryonic diapause.

Proc Natl Acad Sci U S A 2021 03;118(10)

Małopolska Centre of Biotechnology, Jagiellonian University, 30-387 Krakow, Poland;

Embryonic diapause (ED) is a temporary arrest of an embryo at the blastocyst stage when it waits for the uterine receptivity signal to implant. ED used by over 100 species may also occur in normally "nondiapausing" mammals when the uterine receptivity signal is blocked or delayed. A large number of lipid droplets (LDs) are stored throughout the preimplantation embryo development, but the amount of lipids varies greatly across different mammalian species. Yet, the role of LDs in the mammalian egg and embryo remains unknown. Here, using a mouse model, we provide evidence that LDs play a crucial role in maintaining ED. By mechanical removal of LDs from zygotes, we demonstrated that delipidated embryos are unable to survive during ED. LDs are not essential for normal prompt implantation, without ED. We further demonstrated that with the progression of ED, the amount of intracellular lipid reduces, and composition changes. This decrease in lipid is caused by a switch from carbohydrate metabolism to lipid catabolism in diapausing blastocysts, which also exhibit increased release of exosomes reflecting elevated embryonic signaling to the mother. We have also shown that presence of LDs in the oocytes of various mammals positively corelates with their species-specific length of diapause. Our results reveal the functional role of LDs in embryonic development. These results can help to develop diagnostic techniques and treatment of recurrent implantation failure and will likely ignite further studies in developmental biology and reproductive medicine fields.
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http://dx.doi.org/10.1073/pnas.2018362118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7958255PMC
March 2021

Exposure to Gestational Diabetes Mellitus (GDM) alters DNA methylation in placenta and fetal cord blood.

Diabetes Res Clin Pract 2021 Apr 5;174:108690. Epub 2021 Feb 5.

Genetics and Genome Biology Program, The Hospital for Sick Children, Toronto, Ontario, Canada; Department of Paediatrics, University of Toronto, Toronto, Ontario, Canada; Division of Clinical and Metabolic Genetics, Department of Paediatrics, The Hospital for Sick Children, Toronto, Ontario, Canada; Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada; Institute of Medical Science, School of Graduate Studies, University of Toronto, Toronto, Ontario, Canada. Electronic address:

Background: Fetal exposure to maternal GDM increases offspring risk for adult-onset metabolic syndromes. Epigenetic modifications such as DNA methylation are modulators for fetal metabolic programming and susceptibility to adult-onset disease. This study investigates genome-wide DNA methylation in GDM exposed cord blood and placenta.

Methods: Oral glucose tolerance testing between 24 and 28 weeks of pregnancy was used to determine severity of glucose intolerance. We measured DNA methylation (DNAm) using the Illumina Infinium 450 K array in 42 fetal cord blood and 36 placenta samples.

Results: We identified 662 and 99 CpG sites in GDM placenta and cord blood, respectively at p-value <0.01 and a methylation difference >5% after adjustment for confounders. Annotated sites for AHRR and PTPRN2 were common to cord blood and placenta. Adding published GDM cord blood DNAm data we segregated patients based on treatment (Diet Only vs. +Insulin) and identified altered CpG sites to be unique to each GDM treatment group.

Conclusion: Consistency in findings with other studies provides evidence for the role of DNAm in placental and fetal responses to hyperglycemia. However, segregating DNAm analysis of GDM samples based on treatment may help delineate whether observed DNAm alterations are reflective of adaptive responses or treatment effects in utero.
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http://dx.doi.org/10.1016/j.diabres.2021.108690DOI Listing
April 2021

A biallelic variant in CLRN2 causes non-syndromic hearing loss in humans.

Hum Genet 2021 Jun 26;140(6):915-931. Epub 2021 Jan 26.

Department of Genetics, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran.

Deafness, the most frequent sensory deficit in humans, is extremely heterogeneous with hundreds of genes involved. Clinical and genetic analyses of an extended consanguineous family with pre-lingual, moderate-to-profound autosomal recessive sensorineural hearing loss, allowed us to identify CLRN2, encoding a tetraspan protein, as a new deafness gene. Homozygosity mapping followed by exome sequencing identified a 14.96 Mb locus on chromosome 4p15.32p15.1 containing a likely pathogenic missense variant in CLRN2 (c.494C > A, NM_001079827.2) segregating with the disease. Using in vitro RNA splicing analysis, we show that the CLRN2 c.494C > A variant leads to two events: (1) the substitution of a highly conserved threonine (uncharged amino acid) to lysine (charged amino acid) at position 165, p.(Thr165Lys), and (2) aberrant splicing, with the retention of intron 2 resulting in a stop codon after 26 additional amino acids, p.(Gly146Lysfs*26). Expression studies and phenotyping of newly produced zebrafish and mouse models deficient for clarin 2 further confirm that clarin 2, expressed in the inner ear hair cells, is essential for normal organization and maintenance of the auditory hair bundles, and for hearing function. Together, our findings identify CLRN2 as a new deafness gene, which will impact future diagnosis and treatment for deaf patients.
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http://dx.doi.org/10.1007/s00439-020-02254-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8099798PMC
June 2021

A genome-wide transcriptomic analysis of embryos fathered by obese males in a murine model of diet-induced obesity.

Sci Rep 2021 01 21;11(1):1979. Epub 2021 Jan 21.

Institute of Human Genetics, Julius Maximilians University, 97074, Würzburg, Germany.

Paternal obesity is known to have a negative impact on the male's reproductive health as well as the health of his offspring. Although epigenetic mechanisms have been implicated in the non-genetic transmission of acquired traits, the effect of paternal obesity on gene expression in the preimplantation embryo has not been fully studied. To this end, we investigated whether paternal obesity is associated with gene expression changes in eight-cell stage embryos fathered by males on a high-fat diet. We used single embryo RNA-seq to compare the gene expression profile of embryos generated by males on a high fat (HFD) versus control (CD) diet. This analysis revealed significant upregulation of the Samd4b and Gata6 gene in embryos in response to a paternal HFD. Furthermore, we could show a significant increase in expression of both Gata6 and Samd4b during differentiation of stromal vascular cells into mature adipocytes. These findings suggest that paternal obesity may induce changes in the male germ cells which are associated with the gene expression changes in the resulting preimplantation embryos.
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http://dx.doi.org/10.1038/s41598-021-81226-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820458PMC
January 2021

SCUBE3 loss-of-function causes a recognizable recessive developmental disorder due to defective bone morphogenetic protein signaling.

Am J Hum Genet 2021 01 11;108(1):115-133. Epub 2020 Dec 11.

Sheba Cancer Research Center, Sheba Medical Center, 52621 Tel-Hashomer, Israel; Wohl Institute for Translational Medicine, Sheba Medical Center, 52621 Tel-Hashomer, Israel.

Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) is a member of a small family of multifunctional cell surface-anchored glycoproteins functioning as co-receptors for a variety of growth factors. Here we report that bi-allelic inactivating variants in SCUBE3 have pleiotropic consequences on development and cause a previously unrecognized syndromic disorder. Eighteen affected individuals from nine unrelated families showed a consistent phenotype characterized by reduced growth, skeletal features, distinctive craniofacial appearance, and dental anomalies. In vitro functional validation studies demonstrated a variable impact of disease-causing variants on transcript processing, protein secretion and function, and their dysregulating effect on bone morphogenetic protein (BMP) signaling. We show that SCUBE3 acts as a BMP2/BMP4 co-receptor, recruits the BMP receptor complexes into raft microdomains, and positively modulates signaling possibly by augmenting the specific interactions between BMPs and BMP type I receptors. Scube3 mice showed craniofacial and dental defects, reduced body size, and defective endochondral bone growth due to impaired BMP-mediated chondrogenesis and osteogenesis, recapitulating the human disorder. Our findings identify a human disease caused by defective function of a member of the SCUBE family, and link SCUBE3 to processes controlling growth, morphogenesis, and bone and teeth development through modulation of BMP signaling.
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http://dx.doi.org/10.1016/j.ajhg.2020.11.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820739PMC
January 2021

Genetic Spectrum of Syndromic and Non-Syndromic Hearing Loss in Pakistani Families.

Genes (Basel) 2020 11 11;11(11). Epub 2020 Nov 11.

Institute of Human Genetics, Julius Maximilians University Würzburg, 97074 Würzburg, Germany.

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 , , , , , , and , with an overall resolve rate of 61.9%. and 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.
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http://dx.doi.org/10.3390/genes11111329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7709052PMC
November 2020

Correction to: Molecular karyotyping and gene expression analysis in childhood cancer patients.

J Mol Med (Berl) 2020 Nov;98(11):1657

Department of Radiation Oncology and Radiation Therapy, University Medical Centre, Johannes Gutenberg University Mainz, Obere Zahlbacher Str. 63, 55131, Mainz, Germany.

The correct Author names are presented in this paper.
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http://dx.doi.org/10.1007/s00109-020-01986-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852910PMC
November 2020

The First Inherited Retinal Disease Registry in Iran: Research Protocol and Results of a Pilot Study.

Arch Iran Med 2020 07 1;23(7):445-454. Epub 2020 Jul 1.

Ophthalmic Research Center, Research Institute for Ophthalmology and Vision Science, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

Background: To describe the protocol for developing a national inherited retinal disease (IRD) registry in Iran and present its initial report.

Methods: This community-based participatory research was approved by the Ministry of Health and Medical Education of Iran in 2016. To provide the minimum data set (MDS), several focus group meetings were held. The final MDS was handed over to an engineering team to develop a web-based software. In the pilot phase, the software was set up in two referral centers in Iran. Final IRD diagnosis was made based on clinical manifestations and genetic findings. Ultimately, patient registration was done based on all clinical and non-clinical manifestations.

Results: Initially, a total of 151 data elements were approved with Delphi technique. The registry software went live at www. IRDReg.org based on DHIS2 open source license agreement since February 2016. So far, a total of 1001 patients have been registered with a mean age of 32.41±15.60 years (range, 3 months to 74 years). The majority of the registered patients had retinitis pigmentosa (42%, 95% CI: 38.9% to 45%). Genetic testing was done for approximately 20% of the registered individuals.

Conclusion: Our study shows successful web-based software design and data collection as a proof of concept for the first IRD registry in Iran. Multicenter integration of the IRD registry in medical centers throughout the country is well underway as planned. These data will assist researchers to rapidly access information about the distribution and genetic patterns of this disease.
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http://dx.doi.org/10.34172/aim.2020.41DOI Listing
July 2020

Increasing methylation of sperm rDNA and other repetitive elements in the aging male mammalian germline.

Aging Cell 2020 08 1;19(8):e13181. Epub 2020 Jul 1.

Institute of Human Genetics, Julius Maximilians University, Würzburg, Germany.

In somatic cells/tissues, methylation of ribosomal DNA (rDNA) increases with age and age-related pathologies, which has a direct impact on the regulation of nucleolar activity and cellular metabolism. Here, we used bisulfite pyrosequencing and show that methylation of the rDNA transcription unit including upstream control element (UCE), core promoter, 18S rDNA, and 28S rDNA in human sperm also significantly increases with donor's age. This positive correlation between sperm rDNA methylation and biological age is evolutionarily conserved among mammals with widely different life spans such as humans, marmoset, bovine, and mouse. Similar to the tandemly repeated rDNA, methylation of human α-satellite and interspersed LINE1 repeats, marmoset α-satellite, bovine alpha- and testis satellite I, mouse minor and major satellite, and LINE1-T repeats increases in the aging male germline, probably related to their sperm histone packaging. Deep bisulfite sequencing of single rDNA molecules in human sperm revealed that methylation does not only depend on donor's age, but also depend on the region and sequence context (A vs. G alleles). Both average rDNA methylation of all analyzed DNA molecules and the number of fully (>50%) methylated alleles, which are thought to be epigenetically silenced, increase with donor's age. All analyzed CpGs in the sperm rDNA transcription unit show comparable age-related methylation changes. Unlike other epigenetic aging markers, the rDNA clock appears to operate in similar ways in germline and soma in different mammalian species. We propose that sperm rDNA methylation, directly or indirectly, influences nucleolar formation and developmental potential in the early embryo.
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http://dx.doi.org/10.1111/acel.13181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431825PMC
August 2020

Molecular karyotyping and gene expression analysis in childhood cancer patients.

J Mol Med (Berl) 2020 08 23;98(8):1107-1123. Epub 2020 Jun 23.

Department of Radiation Oncology and Radiation Therapy, University Medical Centre, Johannes Gutenberg University Mainz, Obere Zahlbacher Str. 63, 55131, Mainz, Germany.

The genetic etiology of sporadic childhood cancer cases remains unclear. We recruited a cohort of 20 patients who survived a childhood malignancy and then developed a second primary cancer (2N), and 20 carefully matched patients who survived a childhood cancer without developing a second malignancy (1N). Twenty matched cancer-free (0N) and additional 1000 (0N) GHS participants served as controls. Aiming to identify new candidate loci for cancer predisposition, we compared the genome-wide DNA copy number variations (CNV) with the RNA-expression data obtained after in vitro irradiation of primary fibroblasts. In 2N patients, we detected a total of 142 genes affected by CNV. A total of 53 genes of these were not altered in controls. Six genes (POLR3F, SEC23B, ZNF133, C16orf45, RRN3, and NTAN1) that we found to be overexpressed after irradiation were also duplicated in the genome of the 2N patients. For the 1N collective, 185 genes were affected by CNV and 38 of these genes were not altered in controls. Five genes (ZCWPW2, SYNCRIP, DHX30, DHRS4L2, and THSD1) were located in duplicated genomic regions and exhibited altered RNA expression after irradiation. One gene (ABCC6) was partially duplicated in one 1N and one 2N patient. Analysis of methylation levels of THSD1 and GSTT2 genes which were detected in duplicated regions and are frequently aberrantly methylated in cancer showed no changes in patient's fibroblasts. In summary, we describe rare and radiation-sensitive genes affected by CNV in childhood sporadic cancer cases, which may have an impact on cancer development. KEY MESSAGES: • Rare CNV's may have an impact on cancer development in sporadic, non-familial, non-syndromic childhood cancer cases. • In our cohort, each patient displayed a unique pattern of cancer-related gene CNVs, and only few cases shared similar CNV. • Genes that are transcriptionally regulated after radiation can be located in CNVs in cancer patients and controls. • THSD1 and GSTT2 methylation is not altered by CNV.
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http://dx.doi.org/10.1007/s00109-020-01937-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7769790PMC
August 2020

A novel missense variant in MYO3A is associated with autosomal dominant high-frequency hearing loss in a German family.

Mol Genet Genomic Med 2020 08 10;8(8):e1343. Epub 2020 Jun 10.

Institute of Human Genetics, Julius Maximilians University, Würzburg, Germany.

Background: MYO3A, encoding the myosin IIIA protein, is associated with autosomal recessive and autosomal dominant nonsyndromic hearing loss. To date, only two missense variants located in the motor-head domain of MYO3A have been described in autosomal dominant families with progressive, mild-to-profound sensorineural hearing loss. These variants alter the ATPase activity of myosin IIIA.

Methods: Exome sequencing of a proband from a three-generation German family with prelingual, moderate-to-profound, high-frequency hearing loss was performed. Segregation analysis confirmed a dominant inheritance pattern. Regression analysis of mean hearing level thresholds per individual and ear was performed at high-, mid-, and low-frequencies.

Results: A novel heterozygous missense variant c.716T>C, p.(Leu239Pro) in the kinase domain of MYO3A was identified that is predicted in silico as disease causing. High-frequency, progressive hearing loss was identified.

Conclusion: Correlation analysis of pure-tone hearing thresholds revealed progressive hearing loss, especially in the high-frequencies. In the present study, we report the first dominant likely pathogenic variant in MYO3A in a European family and further support MYO3A as an autosomal dominant hearing loss gene.
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http://dx.doi.org/10.1002/mgg3.1343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7434730PMC
August 2020

Parental metabolic syndrome epigenetically reprograms offspring hepatic lipid metabolism in mice.

J Clin Invest 2020 05;130(5):2391-2407

Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, Massachusetts, USA.

The prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing worldwide. Although gene-environment interactions have been implicated in the etiology of several disorders, the impact of paternal and/or maternal metabolic syndrome on the clinical phenotypes of offspring and the underlying genetic and epigenetic contributors of NAFLD have not been fully explored. To this end, we used the liver-specific insulin receptor knockout (LIRKO) mouse, a unique nondietary model manifesting 3 hallmarks that confer high risk for the development of NAFLD: hyperglycemia, insulin resistance, and dyslipidemia. We report that parental metabolic syndrome epigenetically reprograms members of the TGF-β family, including neuronal regeneration-related protein (NREP) and growth differentiation factor 15 (GDF15). NREP and GDF15 modulate the expression of several genes involved in the regulation of hepatic lipid metabolism. In particular, NREP downregulation increases the protein abundance of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and ATP-citrate lyase (ACLY) in a TGF-β receptor/PI3K/protein kinase B-dependent manner, to regulate hepatic acetyl-CoA and cholesterol synthesis. Reduced hepatic expression of NREP in patients with NAFLD and substantial correlations between low serum NREP levels and the presence of steatosis and nonalcoholic steatohepatitis highlight the clinical translational relevance of our findings in the context of recent preclinical trials implicating ACLY in NAFLD progression.
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http://dx.doi.org/10.1172/JCI127502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7190992PMC
May 2020

Biallelic mutations cause a novel syndromal disease due to hampered cellular collagen secretion.

Elife 2020 02 26;9. Epub 2020 Feb 26.

Institute of Human Genetics, Julius Maximilians University Würzburg, Würzburg, Germany.

The transport and Golgi organization 1 (TANGO1) proteins play pivotal roles in the secretory pathway. Full length TANGO1 is a transmembrane protein localised at endoplasmic reticulum (ER) exit sites, where it binds bulky cargo within the ER lumen and recruits membranes from the ER Golgi intermediate compartment to create an exit route for their export. Here we report the first TANGO1-associated syndrome in humans. A synonymous substitution that results in exon eight skipping in most mRNA molecules, ultimately leading to a truncated TANGO1 protein was identified as disease-causing mutation. The four homozygously affected sons of a consanguineous family display severe dentinogenesis imperfecta, short stature, various skeletal abnormalities, insulin-dependent diabetes mellitus, sensorineural hearing loss, and mild intellectual disability. Functional studies in HeLa and U2OS cells revealed that the corresponding truncated TANGO1 protein is dispersed in the ER and its expression in cells with intact endogenous TANGO1 impairs cellular collagen I secretion.
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http://dx.doi.org/10.7554/eLife.51319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7062462PMC
February 2020

Small fish, big prospects: using zebrafish to unravel the mechanisms of hereditary hearing loss.

Hear Res 2020 11 6;397:107906. Epub 2020 Feb 6.

Genes & Human Disease Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, United States. Electronic address:

Over the past decade, advancements in high-throughput sequencing have greatly enhanced our knowledge of the mutational signatures responsible for hereditary hearing loss. In its present state, the field has a largely uncensored view of protein coding changes in a growing number of genes that have been associated with hereditary hearing loss, and many more that have been proposed as candidate genes. Sequencing data can now be generated using methods that have become widespread and affordable. The greatest hurdles facing the field concern functional validation of uncharacterized genes and rapid application to human diseases, including hearing and balance disorders. To date, over 30 hearing-related disease models exist in zebrafish. New genome editing technologies, including CRISPR/Cas9 will accelerate the functional validation of hearing loss genes and variants in zebrafish. Here, we discuss current progress in the field and recent advances in genome editing approaches.
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http://dx.doi.org/10.1016/j.heares.2020.107906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415493PMC
November 2020

In Vitro Fertilization Technology and Child Health.

Dtsch Arztebl Int 2020 01;117(3):23-30

Department of Gynecologic Endocrinology and Reproductive Medicine, University Clinic of Obstetrics and Gynecology, Inselspital Bern, Bern, Switzerland; Institute of Human Genetics, Julius Maximilians University, Würzburg, Germany.

Background: Just under 3% of children in Germany, and approximately 6% of children in some other countries, such as Denmark, are now being conceived with the aid of in vitro fertilization (IVF) technology. Alongside the increased risk of organ malformation, there is now evidence for functional abnormalities due to epigenetic modifications.

Methods: This review is based on pertinent publications retrieved by a literature search on currently known associations of IVF therapy with malformations and functional abnormalities. The potential implications for the treatment of infertility are discussed.

Results: The risk of congenital malformations is approximately one-third higher in children conceived with the aid of IVF technology than in other children; specifically, there is an odds ratio (OR) of 1.29 (95% confidence interval, [1.03; 1.60]) for cardiac malformations, and there is a relative risk (RR) of 1.35 ([1.12; 1.64]) for musculo- skeletal malformations and 1.58 ([1.28; 1.94]) for genitourinary malformations. The risks of preterm birth and low birth weight are, respectively, 1.7 and 1.5 times higher in IVF singleton pregnancies than in non-IVF pregnancies. Cardiovascular changes are the main type of functional disturbance. Some of the risks associated with IVF have decreased in recent years. An association has been revealed between cardiovascular abnormalities and epigenetic modifications; the causes are thought to include not only maternal and paternal factors, but also the IVF techniques that are used. A modification of IVF therapies might lower the risks, but might also lower the success rate.

Conclusion: For the well-being of the children to be conceived, IVF therapy should hat cannot be treated by any other means, as the precise causes of the risks of IVF to child health are unclear.
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http://dx.doi.org/10.3238/arztebl.2020.0023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026576PMC
January 2020

Lessons for aging from Werner syndrome epigenetics.

Aging (Albany NY) 2020 02 5;12(3):2022-2023. Epub 2020 Feb 5.

Institute of Human Genetics, Julius-Maximilians University, 97074 Wuerzburg, Germany.

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http://dx.doi.org/10.18632/aging.102829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7041779PMC
February 2020

Novel Loss-of-Function Variants in are Associated with Recessive Sensorineural Hearing Loss in Iranian and Pakistani Patients.

Int J Mol Sci 2020 Jan 2;21(1). Epub 2020 Jan 2.

Institute of Human Genetics, Julius Maximilians University, 97074 Würzburg, Germany.

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 , 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 is associated with a rare form of hereditary hearing loss.
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http://dx.doi.org/10.3390/ijms21010311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6982189PMC
January 2020

Methylomic profiling in trisomy 21 identifies cognition- and Alzheimer's disease-related dysregulation.

Clin Epigenetics 2019 12 16;11(1):195. Epub 2019 Dec 16.

Institut Jérôme Lejeune, CRB BioJeL, 37 rue des Volontaires, Paris, France.

Background: Trisomy 21 (T21) is associated with intellectual disability that ranges from mild to profound with an average intellectual quotient of around 50. Furthermore, T21 patients have a high risk of developing Alzheimer's disease (AD) early in life, characterized by the presence of senile plaques of amyloid protein and neurofibrillary tangles, leading to neuronal loss and cognitive decline. We postulate that epigenetic factors contribute to the observed variability in intellectual disability, as well as at the level of neurodegeneration seen in T21 individuals.

Materials And Methods: A genome-wide DNA methylation study was performed using Illumina Infinium® MethylationEPIC BeadChips on whole blood DNA of 3 male T21 patients with low IQ, 8 T21 patients with high IQ (4 males and 4 females), and 21 age- and sex-matched control samples (12 males and 9 females) in order to determine whether DNA methylation alterations could help explain variation in cognitive impairment between individuals with T21. In view of the increased risk of developing AD in T21 individuals, we additionally investigated the T21-associated sites in published blood DNA methylation data from the AgeCoDe cohort (German study on Ageing, Cognition, and Dementia). AgeCoDe represents a prospective longitudinal study including non-demented individuals at baseline of which a part develops AD dementia at follow-up.

Results: Two thousand seven hundred sixteen differentially methylated sites and regions discriminating T21 and healthy individuals were identified. In the T21 high and low IQ comparison, a single CpG located in the promoter of PELI1 was differentially methylated after multiple testing adjustment. For the same contrast, 69 differentially methylated regions were identified. Performing a targeted association analysis for the significant T21-associated CpG sites in the AgeCoDe cohort, we found that 9 showed significant methylation differences related to AD dementia, including one in the ADAM10 gene. This gene has previously been shown to play a role in the prevention of amyloid plaque formation in the brain.

Conclusion: The differentially methylated regions may help understand the interaction between methylation alterations and cognitive function. In addition, ADAM10 might be a valuable blood-based biomarker for at least the early detection of AD.
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http://dx.doi.org/10.1186/s13148-019-0787-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6916110PMC
December 2019

Memory CD4+ T cells lacking expression of CCR7 promote pro-inflammatory cytokine production in patients with diffuse cutaneous systemic sclerosis.

Eur J Dermatol 2019 Oct;29(5):468-476

Department of Pediatrics, University Hospital Würzburg, Würzburg, Germany.

Systemic sclerosis (SSc) is a predominantly T-cell-mediated autoimmune disorder with a characteristic sequence of Th1 and Th2 inflammation resulting in fibrosis. The contribution of differentiated memory T-cell subpopulations and methylation of CpG regions of Th1- or Th2-specific transcription factor genes on the inflammatory cytokine signature in SSc is not well understood. The study aimed to investigate phenotypic differentiation, the cytokine signature, sensitivity of memory T cells to in vitro suppression by autologous regulatory T cells (Tregs), and methylation of Th1- and Th2-specific transcription factor genes in patients with limited (lcSSc) and diffuse cutaneous SSc (dcSSc) compared to healthy donors (HD). Phenotype/intracellular cytokine production and methylation of Th1- and Th2-specific transcription factor genes were determined by flow cytometry and epigenetic analysis, respectively, and compared between patients with lcSSc, dcSSc and HD. Discrimination of CD4+ T cells that lack CCR7 expression revealed that CCR7- CD4+ memory T cells and effectors are producers of intracellular TNFα, IL-13 and IL-4, particularly in dcSSc. A proportional increase in CCR7- memory T cells was demonstrated by SSc-derived CD4+ T-cells after insufficient suppression by Tregs. A higher level of methylation of GATA3 or STAT4 (Th2- and Th1-specific transcription factor genes, respectively) was observed in dcSSc. An abundance of specific CD4+ memory T-cell subpopulations strongly contributes to the production of pro-inflammatory cytokines in dcSSc. Our results suggest that therapeutic concepts should focus more intensively on the memory phenotype to control T cell-mediated inflammation in SSc patients.
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http://dx.doi.org/10.1684/ejd.2019.3645DOI Listing
October 2019

Homozygous Null TBX4 Mutations Lead to Posterior Amelia with Pelvic and Pulmonary Hypoplasia.

Am J Hum Genet 2019 12 21;105(6):1294-1301. Epub 2019 Nov 21.

Institute of Medical Biology, Agency for Science, Technology, and Research, 8A Biomedical Grove, Singapore 138648, Republic of Singapore; Institute of Molecular and Cell Biology, Agency for Science, Technology, and Research, 61 Biopolis Drive, Singapore 138673, Republic of Singapore; Department of Medical Genetics, Koç University, School of Medicine, 34010 Topkapı, Istanbul, Turkey. Electronic address:

The development of hindlimbs in tetrapod species relies specifically on the transcription factor TBX4. In humans, heterozygous loss-of-function TBX4 mutations cause dominant small patella syndrome (SPS) due to haploinsufficiency. Here, we characterize a striking clinical entity in four fetuses with complete posterior amelia with pelvis and pulmonary hypoplasia (PAPPA). Through exome sequencing, we find that PAPPA syndrome is caused by homozygous TBX4 inactivating mutations during embryogenesis in humans. In two consanguineous couples, we uncover distinct germline TBX4 coding mutations, p.Tyr113 and p.Tyr127Asn, that segregated with SPS in heterozygous parents and with posterior amelia with pelvis and pulmonary hypoplasia syndrome (PAPPAS) in one available homozygous fetus. A complete absence of TBX4 transcripts in this proband with biallelic p.Tyr113 stop-gain mutations revealed nonsense-mediated decay of the endogenous mRNA. CRISPR/Cas9-mediated TBX4 deletion in Xenopus embryos confirmed its restricted role during leg development. We conclude that SPS and PAPPAS are allelic diseases of TBX4 deficiency and that TBX4 is an essential transcription factor for organogenesis of the lungs, pelvis, and hindlimbs in humans.
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http://dx.doi.org/10.1016/j.ajhg.2019.10.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904794PMC
December 2019

Genome-wide DNA methylation analysis of colorectal adenomas with and without recurrence reveals an association between cytosine-phosphate-guanine methylation and histological subtypes.

Genes Chromosomes Cancer 2019 11 10;58(11):783-797. Epub 2019 Aug 10.

Institute of Pathology, University Medical Center Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany.

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.
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http://dx.doi.org/10.1002/gcc.22787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8268761PMC
November 2019

Exome-wide copy number variation analysis identifies a COL9A1 in frame deletion that is associated with hearing loss.

Eur J Med Genet 2019 Oct 14;62(10):103724. Epub 2019 Jul 14.

Institute of Human Genetics, Julius Maximilians University, Würzburg, Germany; Department of Otorhinolaryngology-Head and Neck Surgery, Tübingen Hearing Research Centre, Eberhard Karls University Tübingen, Tübingen, Germany. Electronic address:

Pathogenic variants in COL9A1 are primarily associated with autosomal recessive Stickler syndrome. Patients with COL9A1-associated Stickler syndrome (STL) present hearing loss (HL), ophthalmic manifestations and skeletal abnormalities. However, the clinical spectrum of patients with COL9A1 variants can also include multiple epiphyseal dysplasia, as well as non-syndromic HL that was observed in one previously reported proband. Exome sequencing was performed on the genomic DNA of an Iranian patient and his affected brother who both report non-syndromic HL. A 44.6 kb homozygous in-frame deletion spanning exons 6 to 33 of COL9A1 was detected via exome-based copy number variation analysis. The deleted exons were confirmed by PCR in the patient and his affected brother, who both have non-syndromic HL. Segregation analysis via qPCR confirmed the parents as heterozygous deletion carriers. Breakpoint analysis mapped the homozygous deletion spanning introns 5 to 33 (g.70,948,188_70,997,277del, NM_001851.4(COL9A1):c.697-3754_2112+769del, p.(Phe233_Ser704del), with an additional 67 bp of inserted intronic sequence that may have originated due to a fork stalling and template switching/microhomology-mediated break-induced replication (FoSTeS/MMBIR) mechanism. This mechanism has not been previously implicated in HL or STL. This is also the first reported copy number variation in COL9A1 that was identified through an exome data set in an Iranian family with apparent non-syndromic HL. The present study emphasizes the importance of exome-wide copy number variation analysis in molecular diagnosis and provides supporting evidence to associate COL9A1 with autosomal recessive non-syndromic HL.
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http://dx.doi.org/10.1016/j.ejmg.2019.103724DOI Listing
October 2019

Epigenetic signatures of Werner syndrome occur early in life and are distinct from normal epigenetic aging processes.

Aging Cell 2019 10 1;18(5):e12995. Epub 2019 Jul 1.

Institute of Human Genetics, Julius Maximilians University, Würzburg, Germany.

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.
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http://dx.doi.org/10.1111/acel.12995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6718529PMC
October 2019
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