DOCSLIB.ORG

Efficient Mapping of Transgene Integration Sites and Local Structural

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Efficient Mapping of Transgene Integration Sites and Local Structural Published online 4 January 2017 Nucleic Acids Research, 2017, Vol. 45, No. 8 e62 doi: 10.1093/nar/gkw1329 Efficient mapping of transgene integration sites and local structural changes in Cre transgenic mice using targeted locus amplification Carol Cain-Hom1, Erik Splinter2, Max van Min2, Marieke Simonis2, Monique van de Heijning2, Maria Martinez1, Vida Asghari1, J. Colin Cox1,* and Søren Warming3,* 1Department of Transgenic Technology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA, 2Cergentis BV, Yalelaan 62, 3584 CM Utrecht, the Netherlands and 3Department of Molecular Biology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA Received October 7, 2016; Revised December 13, 2016; Editorial Decision December 19, 2016; Accepted December 20, 2016 ABSTRACT function (1–3) and hundreds of Cre ‘deleter’ lines are avail- / able to the mouse community. Cell-type specific expression Cre LoxP technology is widely used in the field of of Cre allows for specific deletion of a gene of interest by / mouse genetics for spatial and or temporal regula- the use of a ‘conditional knock-out’ (CKO) allele of that tion of gene function. For Cre lines generated via gene (2). Typically, for a conditional allele a critical exon(s) pronuclear microinjection of a Cre transgene con- is flanked by two loxP sites (‘floxed’) and in the cells where struct, the integration site is random and in most Cre is expressed, the floxed exon(s) is removed, resulting in cases not known. Integration of a transgene can dis- a deletion, or knock-out, allele. Cre deleter lines are gener- rupt an endogenous gene, potentially interfering with ated either by targeted knock-in of the Cre cDNA into an interpretation of the phenotype. In addition, knowl- endogenous locus or by pronuclear microinjection of a Cre edge of where the transgene is integrated is impor- transgene driven by a cell-type specific promoter. For the tant for planning of crosses between animals carry- latter, the integration site is random and in most cases not known. Knowledge of where the transgene is integrated is ing a conditional allele and a given Cre allele in case important for planning of crosses between animals carrying the alleles are on the same chromosome. We have a conditional allele and a given Cre allele in case the alle- used targeted locus amplification (TLA) to efficiently les are on the same chromosome. This becomes increasingly map the transgene location in seven previously pub- important in complex crosses with multiple conditional al- lished Cre and CreERT2 transgenic lines. In all lines, leles, as some combinations of alleles might not be possible. transgene insertion was associated with structural Importantly, integration of a transgene can disrupt an en- changes of variable complexity, illustrating the im- dogenous gene, potentially interfering with interpretation portance of testing for rearrangements around the of the transgenic phenotype (4–6) or preventing the gen- integration site. In all seven lines the exact integra- eration of homozygous transgenic animals due to embry- tion site and breakpoint sequences were identified. onic lethality when the transgene is bred to homozygosity. Our methods, data and genotyping assays can be Transgenes often integrate as a multicopy concatemer (7) and in the absence of integration site data, hemi- and ho- used as a resource for the mouse community and mozygous animals have to be distinguished by copy number our results illustrate the power of the TLA method variation (CNV) analysis which involves quantitative poly- to not only efficiently map the integration site of any merase chain reaction (PCR) and reference DNA with a transgene, but also provide additional information re- known copy number. The resolution and reliability of CNV garding the transgene integration events. analysis for accurate genotyping decreases as copy num- ber increases. Therefore, another important and practical use of knowing the exact transgene insertion site is that ef- INTRODUCTION ficient, copy-number independent and locus-specific geno- Cre/LoxP technology is widely used in the field of mouse typing assays can be developed. For large transgenic ani- genetics for spatial and/or temporal regulation of gene mal facilities, where a large number of genotyping assays are *To whom correspondence should be addressed. Tel: +1 650 467 8027; Email: [email protected] Correspondence may also be addressed to J. Colin Cox. Tel: +1 530 753 5883; Email: [email protected] C The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] e62 Nucleic Acids Research, 2017, Vol. 45, No. 8 PAGE 2 OF 9 performed, automated genotyping platforms and robust as- selective amplification of DNA and results in the amplifica- says are necessary (8). CNV analysis is more labor-intensive tion of >100 kb of sequence information at either end of a than real-time PCR assays due to the need for an internal primer pair complementary to a short (trans-) gene specific copy number standard and the additional requirement for sequence. As such, TLA-based targeted sequencing enables replicates to ensure accurate copy-number calling (9). Al- targeted complete sequencing of loci of interest and detec- ternatively, if the insertion site is known, an assay can be tion of all single nucleotide variations (SNVs) and struc- designed for the wild-type DNA and for the insertion, al- tural variants. Here we describe the use of TLA to efficiently lowing for analysis without having to calculate copy number map the transgene location in seven previously published in order to determine zygosity. Thus the characterization of Cre and CreERT2 transgenic lines. These seven mouse lines transgene integration sites is important both to know if any are all widely used by the mouse community. For exam- rearrangements have occurred in the endogenous sequence ple, see http://www.informatics.jax.org/home/recombinase of the integration site and to enable automated genotyping for an up-to-date list of references for each transgenic line. of as many of the Cre transgenic lines as possible. With very limited sequence data available for the transgenes The most commonly used method for identification of used to generate these lines, Cre transgenic lines provided transgene insertion sites is based on inverse PCR (iPCR) an ideal test-case for the power of the TLA approach to (10,11). This method relies on knowledge of appropriate transgene mapping. Because the mouse lines all have the restriction sites located in the transgene and on sequence Cre sequence in common, we designed primers on the Cre information to design appropriate primers for the iPCR. sequence and these primers could then be used in the anal- Furthermore, iPCR works best if the number of transgene ysis for all Cre lines. After mapping the exact integration copies is low as selective amplification of the transgene con- sites and identifying structural changes, we used the infor- catemer reduces the chance of identifying a PCR prod- mation to design quantitative PCR genotyping assays that uct containing flanking genomic sequence12 ( ). An alter- distinguish wild-type, hemizygous and homozygous ani- native method for identification of insertion sites, ‘Splink- mals for four of the lines. Our data illustrates the power erette’, was first developed for the cloning of retroviral in- of the TLA method to sequence transgenes and efficiently tegration sites (13). Just as for iPCR, Splinkerette is best map their integration sites with very limited requirement for suited for single or low copy integrations. While iPCR and prior knowledge of the transgene sequence. Furthermore, Splinkerette can be used to identify the genomic/transgene our data illustrates the power of TLA to identify structural junction, both methods rely on prior knowledge of the changes occurring at the site of transgene integration. end(s) of integrated transgene sequences and on whether the breakpoint sequence is suited for PCR amplification. In MATERIALS AND METHODS addition, neither method provides any information about Mouse strains structural changes at the integration site. Although both iPCR and Splinkerette methods now take advantage of The Cre and CreERT2 transgenic strains used in next-generation sequencing (NGS) (for example, see (14)), this study were licensed and obtained from different NGS technology alternatives to iPCR and Splinkerette for sources and then maintained at Genentech. ACTB-Cre: transgene mapping now include whole genome sequencing FVB/N-Tg(ACTB-cre)2Mrt/J(19). Obtained from (15,16) and sequence capture followed by NGS (17). Whole the Jackson laboratory (stock no. 003376) and back- genome sequencing analyses are not suited for the detailed crossed until congenic on C57BL/6N at Genentech. analysis of the transgene sequence since most of the gener- BEST1-Cre: C57BL/6-Tg(BEST1-cre)1Jdun/J(20). Ob- ated sequencing data is uninformative. More importantly, in tained from the Jackson laboratory (stock no. 017557). hemizygous mice, the generated sequencing data is not only Generated on C57BL/6NCrl, then crossed to and derived from the locus of integration but also from the wild maintained on C57BL/6J. Cdh5-CreERT2: Tg(Cdh5- type locus, obscuring interpretation of structural changes cre/ERT2)CIVE23Mlia (21). Obtained from University specific to the integration site. Moreover, many transgenes of California, LA (UCLA). Generated on C57BL/6J and contain sequences that are homologous to endogenous se- kept on a C57BL/6J background at Genentech. ‘CIVE’: quences and the presence of the endogenous homologs in C57BL/6J, Inducible, VE-cadherin Cre-recombinase whole genome sequencing data precludes sensitive and spe- mouse.
Load more

Recommended publications
  • Genomic Variants Reveal Differential Evolutionary Constraints on Human Transglutaminases and Point Towards Unrecognized Significance of Transglutaminase 2
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by University of Debrecen Electronic Archive RESEARCH ARTICLE Genomic variants reveal differential evolutionary constraints on human transglutaminases and point towards unrecognized significance of transglutaminase 2 Kiruphagaran Thangaraju1, RoÂbert KiraÂly1, MaÂte A. DemeÂny1, JaÂnos AndraÂs MoÂtyaÂn1, a1111111111 Mo nika Fuxreiter1,2, LaÂszlo FeÂsuÈs1,3* a1111111111 a1111111111 1 Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, a1111111111 Debrecen, Hungary, 2 MTA-DE Momentum Laboratory of Protein Dynamics, Faculty of Medicine, University a1111111111 of Debrecen, Debrecen, Hungary, 3 MTA-DE Stem cell, Apoptosis and Genomics Research Group of Hungarian Academy of Sciences, Faculty of Medicine, University of Debrecen, Debrecen, Hungary * [email protected] OPEN ACCESS Abstract Citation: Thangaraju K, KiraÂly R, DemeÂny MA, AndraÂs MoÂtyaÂn J, Fuxreiter M, FeÂsuÈs L (2017) Transglutaminases (TGMs) catalyze Ca2+-dependent transamidation of proteins with speci- Genomic variants reveal differential evolutionary constraints on human transglutaminases and point fied roles in blood clotting (F13a) and in cornification (TGM1, TGM3). The ubiquitous TGM2 towards unrecognized significance of has well described enzymatic and non-enzymatic functions but in-spite of numerous studies transglutaminase 2. PLoS ONE 12(3): e0172189. its physiological function in humans has not been defined. We compared data on non-syn- doi:10.1371/journal.pone.0172189 onymous single nucleotide variations (nsSNVs) and loss-of-function variants on TGM1-7 Editor: Richard L. Eckert, University of Maryland and F13a from the Exome aggregation consortium dataset, and used computational and School of Medicine, UNITED STATES biochemical analysis to reveal the roles of damaging nsSNVs of TGM2.
    [Show full text]
  • TGM6 L517W Is Not a Pathogenic Variant for Spinocerebellar Ataxia Type 35
    ARTICLE OPEN ACCESS TGM6 L517W is not a pathogenic variant for spinocerebellar ataxia type 35 Yanxing Chen, MD, PhD, Dengchang Wu, MD, PhD, Benyan Luo, MD, PhD, Guohua Zhao, MD, PhD, and Correspondence Kang Wang, MD, PhD Dr. Zhao [email protected] Neurol Genet 2020;6:e424. doi:10.1212/NXG.0000000000000424 or Dr. Wang [email protected] Abstract Objective To investigate the pathogenicity of the TGM6 variant for spinocerebellar ataxia 35 (SCA35), which was previously reported to be caused by pathogenic mutations in the gene TGM6. Methods Neurologic assessment and brain MRI were performed to provide detailed description of the phenotype. Whole-exome sequencing and dynamic mutation analysis were performed to identify the genotype. Results The proband, presenting with myoclonic epilepsy, cognitive decline, and ataxia, harbored both the TGM6 p.L517W variant and expanded CAG repeats in gene ATN1. Further analysis of the other living family members in this pedigree revealed that the CAG repeat number was expanded in all the patients and within normal range in all the unaffected family members. However, the TGM6 p.L517W variant was absent in 2 affected family members, but present in 3 healthy individuals. Conclusions The nonsegregation of the TGM6 variant with phenotype does not support this variant as the disease-causing gene in this pedigree, questioning the pathogenicity of TGM6 in SCA35. From the Department of Neurology (Y.C., G.Z.), the Second Affiliated Hospital, School of Medicine, Zhejiang University; and Department of Neurology (D.W., B.L., K.W.), the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.
    [Show full text]
  • Neurologic Outcomes in Friedreich Ataxia: Study of a Single-Site Cohort E415
    Volume 6, Number 3, June 2020 Neurology.org/NG A peer-reviewed clinical and translational neurology open access journal ARTICLE Neurologic outcomes in Friedreich ataxia: Study of a single-site cohort e415 ARTICLE Prevalence of RFC1-mediated spinocerebellar ataxia in a North American ataxia cohort e440 ARTICLE Mutations in the m-AAA proteases AFG3L2 and SPG7 are causing isolated dominant optic atrophy e428 ARTICLE Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy revisited: Genotype-phenotype correlations of all published cases e434 Academy Officers Neurology® is a registered trademark of the American Academy of Neurology (registration valid in the United States). James C. Stevens, MD, FAAN, President Neurology® Genetics (eISSN 2376-7839) is an open access journal published Orly Avitzur, MD, MBA, FAAN, President Elect online for the American Academy of Neurology, 201 Chicago Avenue, Ann H. Tilton, MD, FAAN, Vice President Minneapolis, MN 55415, by Wolters Kluwer Health, Inc. at 14700 Citicorp Drive, Bldg. 3, Hagerstown, MD 21742. Business offices are located at Two Carlayne E. Jackson, MD, FAAN, Secretary Commerce Square, 2001 Market Street, Philadelphia, PA 19103. Production offices are located at 351 West Camden Street, Baltimore, MD 21201-2436. Janis M. Miyasaki, MD, MEd, FRCPC, FAAN, Treasurer © 2020 American Academy of Neurology. Ralph L. Sacco, MD, MS, FAAN, Past President Neurology® Genetics is an official journal of the American Academy of Neurology. Journal website: Neurology.org/ng, AAN website: AAN.com CEO, American Academy of Neurology Copyright and Permission Information: Please go to the journal website (www.neurology.org/ng) and click the Permissions tab for the relevant Mary E.
    [Show full text]
  • Gene Ontology Functional Annotations and Pleiotropy
    Network based analysis of genetic disease associations Sarah Gilman Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy under the Executive Committee of the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2014 © 2013 Sarah Gilman All Rights Reserved ABSTRACT Network based analysis of genetic disease associations Sarah Gilman Despite extensive efforts and many promising early findings, genome-wide association studies have explained only a small fraction of the genetic factors contributing to common human diseases. There are many theories about where this “missing heritability” might lie, but increasingly the prevailing view is that common variants, the target of GWAS, are not solely responsible for susceptibility to common diseases and a substantial portion of human disease risk will be found among rare variants. Relatively new, such variants have not been subject to purifying selection, and therefore may be particularly pertinent for neuropsychiatric disorders and other diseases with greatly reduced fecundity. Recently, several researchers have made great progress towards uncovering the genetics behind autism and schizophrenia. By sequencing families, they have found hundreds of de novo variants occurring only in affected individuals, both large structural copy number variants and single nucleotide variants. Despite studying large cohorts there has been little recurrence among the genes implicated suggesting that many hundreds of genes may underlie these complex phenotypes. The question
    [Show full text]
  • Comprehensive Comparison of Three Commercial Human Whole-Exome
    Asan et al. Genome Biology 2011, 12:R95 http://genomebiology.com/2011/12/9/R95 RESEARCH Open Access Comprehensive comparison of three commercial human whole-exome capture platforms Asan2,3*†,YuXu1†, Hui Jiang1†, Chris Tyler-Smith4†, Yali Xue4, Tao Jiang1, Jiawei Wang1, Mingzhi Wu1, Xiao Liu1, Geng Tian1, Jun Wang1, Jian Wang1, Huangming Yang1* and Xiuqing Zhang1* Abstract Background: Exome sequencing, which allows the global analysis of protein coding sequences in the human genome, has become an effective and affordable approach to detecting causative genetic mutations in diseases. Currently, there are several commercial human exome capture platforms; however, the relative performances of these have not been characterized sufficiently to know which is best for a particular study. Results: We comprehensively compared three platforms: NimbleGen’s Sequence Capture Array and SeqCap EZ, and Agilent’s SureSelect. We assessed their performance in a variety of ways, including number of genes covered and capture efficacy. Differences that may impact on the choice of platform were that Agilent SureSelect covered approximately 1,100 more genes, while NimbleGen provided better flanking sequence capture. Although all three platforms achieved similar capture specificity of targeted regions, the NimbleGen platforms showed better uniformity of coverage and greater genotype sensitivity at 30- to 100-fold sequencing depth. All three platforms showed similar power in exome SNP calling, including medically relevant SNPs. Compared with genotyping and whole-genome sequencing data, the three platforms achieved a similar accuracy of genotype assignment and SNP detection. Importantly, all three platforms showed similar levels of reproducibility, GC bias and reference allele bias. Conclusions: We demonstrate key differences between the three platforms, particularly advantages of solutions over array capture and the importance of a large gene target set.
    [Show full text]
  • Genetic Background of Ataxia in Children Younger Than 5 Years in Finland E444
    Volume 6, Number 4, August 2020 Neurology.org/NG A peer-reviewed clinical and translational neurology open access journal ARTICLE Genetic background of ataxia in children younger than 5 years in Finland e444 ARTICLE Cerebral arteriopathy associated with heterozygous variants in the casitas B-lineage lymphoma gene e448 ARTICLE Somatic SLC35A2 mosaicism correlates with clinical fi ndings in epilepsy brain tissuee460 ARTICLE Synonymous variants associated with Alzheimer disease in multiplex families e450 Academy Officers Neurology® is a registered trademark of the American Academy of Neurology (registration valid in the United States). James C. Stevens, MD, FAAN, President Neurology® Genetics (eISSN 2376-7839) is an open access journal published Orly Avitzur, MD, MBA, FAAN, President Elect online for the American Academy of Neurology, 201 Chicago Avenue, Ann H. Tilton, MD, FAAN, Vice President Minneapolis, MN 55415, by Wolters Kluwer Health, Inc. at 14700 Citicorp Drive, Bldg. 3, Hagerstown, MD 21742. Business offices are located at Two Carlayne E. Jackson, MD, FAAN, Secretary Commerce Square, 2001 Market Street, Philadelphia, PA 19103. Production offices are located at 351 West Camden Street, Baltimore, MD 21201-2436. Janis M. Miyasaki, MD, MEd, FRCPC, FAAN, Treasurer © 2020 American Academy of Neurology. Ralph L. Sacco, MD, MS, FAAN, Past President Neurology® Genetics is an official journal of the American Academy of Neurology. Journal website: Neurology.org/ng, AAN website: AAN.com CEO, American Academy of Neurology Copyright and Permission Information: Please go to the journal website (www.neurology.org/ng) and click the Permissions tab for the relevant Mary E. Post, MBA, CAE article. Alternatively, send an email to [email protected].
    [Show full text]
  • W O 2019/200163 Al 17 October 2019 (17.10.2019) W IPO I PCT
    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property (1) Organization11111111111111111111111I1111111111111i1111liiili International Bureau (10) International Publication Number (43) International Publication Date W O 2019/200163 Al 17 October 2019 (17.10.2019) W IPO I PCT (51) International Patent Classification: DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, C12N15/869 (2006.01) A61P17/00 (2006.01) HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, C07K14/46 (2006.01) KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, (21) International Application Number: MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, PCT/US2019/027079 pM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, (22) International Filing Date: TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. 11 April 2019 (11.04.2019) (84) Designated States (unless otherwise indicated, for every (25) Filing Language: English kind of regionalprotection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, (26)PublicationLanguage: English UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, (30) Priority Data: TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, 62/656,768 12 April 2018 (12.04.2018) US EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, (71) Applicant: KRYSTAL BIOTECH, INC.
    [Show full text]
  • Homozygous Mutations in PXDN Cause Congenital Cataract, Corneal Opacity, and Developmental Glaucoma
    REPORT Homozygous Mutations in PXDN Cause Congenital Cataract, Corneal Opacity, and Developmental Glaucoma Kamron Khan,1,2,11 Adam Rudkin,3,11 David A. Parry,1,11 Kathryn P. Burdon,3 Martin McKibbin,1,2 Clare V. Logan,1 Zakia I.A. Abdelhamed,1,4 James S. Muecke,5 Narcis Fernandez-Fuentes,1 Kate J. Laurie,3 Mike Shires,1 Rhys Fogarty,3 Ian M. Carr,1 James A. Poulter,1 Joanne E. Morgan,1 Moin D. Mohamed,1,6 Hussain Jafri,7 Yasmin Raashid,8 Ngy Meng,9 Horm Piseth,10 Carmel Toomes,1 Robert J. Casson,5 Graham R. Taylor,1 Michael Hammerton,5 Eamonn Sheridan,1 Colin A. Johnson,1 Chris F. Inglehearn,1 Jamie E. Craig,3,11,* and Manir Ali1,11,* Anterior segment dysgenesis describes a group of heterogeneous developmental disorders that affect the anterior chamber of the eye and are associated with an increased risk of glaucoma. Here, we report homozygous mutations in peroxidasin (PXDN) in two consanguineous Pakistani families with congenital cataract-microcornea with mild to moderate corneal opacity and in a consanguineous Cambodian family with developmental glaucoma and severe corneal opacification. These results highlight the diverse ocular phenotypes caused by PXDN mutations, which are likely due to differences in genetic background and environmental factors. Peroxidasin is an extracellular matrix-associated protein with peroxidase catalytic activity, and we confirmed localization of the protein to the cornea and lens epithe- lial layers. Our findings imply that peroxidasin is essential for normal development of the anterior chamber of the eye, where it may have a structural role in supporting cornea and lens architecture as well as an enzymatic role as an antioxidant enzyme in protecting the lens, trabecular meshwork, and cornea against oxidative damage.
    [Show full text]
  • Network Analysis of Potential Risk Genes for Psoriasis Huilin Wang, Wenjun Chen, Jin He, Wenjuan Xu and Jiangwei Liu*
    Wang et al. Hereditas (2021) 158:21 https://doi.org/10.1186/s41065-021-00186-w RESEARCH Open Access Network analysis of potential risk genes for psoriasis Huilin Wang, Wenjun Chen, Jin He, Wenjuan Xu and Jiangwei Liu* Abstract Background: Psoriasis is a complex chronic infammatory skin disease. The aim of this study was to analyze potential risk genes and molecular mechanisms associated with psoriasis. Methods: GSE54456, GSE114286, and GSE121212 were collected from gene expression omnibus (GEO) database. Diferentially expressed genes (DEGs) between psoriasis and controls were screened respectively in three datasets and common DEGs were obtained. The biological role of common DEGs were identifed by enrichment analysis. Hub genes were identifed using protein–protein interaction (PPI) networks and their risk for psoriasis was evalu- ated through logistic regression analysis. Moreover, diferentially methylated positions (DMPs) between psoriasis and controls were obtained in the GSE115797 dataset. Methylation markers were identifed after comparison with the common genes. Results: A total of 118 common DEGs were identifed, which were mainly involved in keratinocyte diferentiation and IL-17 signaling pathway. Through PPI network, we identifed top 10 degrees as hub genes. Among them, high expres- sion of CXCL9 and SPRR1B may be risk factors for psoriasis. In addition, we selected 10 methylation-modifed genes with the higher area under receiver operating characteristic curve (AUC) value as methylation markers. Nomogram showed that TGM6 and S100A9 may be associated with an increased risk of psoriasis. Conclusion: This suggests that immune and infammatory responses are active in keratinocytes of psoriatic skin. CXCL9, SPRR1B, TGM6 and S100A9 may be potential targets for the diagnosis and treatment of psoriasis.
    [Show full text]
  • Transglutaminase 3: the Involvement in Epithelial Differentiation and Cancer
    cells Review Transglutaminase 3: The Involvement in Epithelial Differentiation and Cancer Elina S. Chermnykh * , Elena V. Alpeeva and Ekaterina A. Vorotelyak Koltzov Institute of Developmental Biology Russian Academy of Sciences, 119334 Moscow, Russia; [email protected] (E.V.A.); [email protected] (E.A.V.) * Correspondence: [email protected] Received: 1 June 2020; Accepted: 26 August 2020; Published: 30 August 2020 Abstract: Transglutaminases (TGMs) contribute to the formation of rigid, insoluble macromolecular complexes, which are essential for the epidermis and hair follicles to perform protective and barrier functions against the environment. During differentiation, epidermal keratinocytes undergo structural alterations being transformed into cornified cells, which constitute a highly tough outermost layer of the epidermis, the stratum corneum. Similar processes occur during the hardening of the hair follicle and the hair shaft, which is provided by the enzymatic cross-linking of the structural proteins and keratin intermediate filaments. TGM3, also known as epidermal TGM, is one of the pivotal enzymes responsible for the formation of protein polymers in the epidermis and the hair follicle. Numerous studies have shown that TGM3 is extensively involved in epidermal and hair follicle physiology and pathology. However, the roles of TGM3, its substrates, and its importance for the integument system are not fully understood. Here, we summarize the main advances that have recently been achieved in TGM3 analyses in skin and hair follicle biology and also in understanding the functional role of TGM3 in human tumor pathology as well as the reliability of its prognostic clinical usage as a cancer diagnosis biomarker. This review also focuses on human and murine hair follicle abnormalities connected with TGM3 mutations.
    [Show full text]
  • Molecular Genetic Studies of Recessively Inherited Eye Diseases
    MOLECULAR GENETIC STUDIES OF RECESSIVELY INHERITED EYE DISEASES by SARAH JOYCE A thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Clinical and Experimental Medicine The Medical School University of Birmingham July 2011 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract Cataract is the opacification of the crystalline lens of the eye. Both childhood and later-onset cataracts have been linked with complex genetic factors. Cataracts vary in phenotype and exhibit genetic heterogeneity. They can appear as isolated abnormalities, or as part of a syndrome. During this project, analysis of syndromic and non-syndromic cataract families using genetic linkage studies was undertaken in order to identify the genes involved, using an autozygosity mapping and positional candidate approach. Causative mutations were identified in families with syndromes involving cataracts. The finding of a mutation in CYP27A1 in a family with Cerebrotendinous Xanthomatosis permitted clinical intervention as this is a treatable disorder. A mutation that segregated with disease status in a family with Marinesco Sjogren Syndrome was identified in SIL1. In a family with Knobloch Syndrome, a frameshift mutation in COL18A1 was detected in all affected individuals.
    [Show full text]
  • TGM5 Gene Transglutaminase 5
    TGM5 gene transglutaminase 5 Normal Function The TGM5 gene provides instructions for making an enzyme called transglutaminase 5. This enzyme is found in many of the body's tissues, although it seems to play a particularly important role in the outer layer of skin (the epidermis). In the epidermis, transglutaminase 5 is involved in the formation of the cornified cell envelope, which is a structure that surrounds cells and helps the skin form a protective barrier between the body and its environment. Specifically, transglutaminase 5 forms strong bonds, called cross-links, between the structural proteins that make up the cornified cell envelope. This cross-linking provides strength and stability to the epidermis. Health Conditions Related to Genetic Changes Acral peeling skin syndrome At least 22 mutations in the TGM5 gene have been found to cause acral peeling skin syndrome. This condition is characterized by painless peeling of the top layer of skin that is most apparent on the hands and feet but can also affect the arms and legs. Most of the mutations change single protein building blocks (amino acids) in transglutaminase 5, including the most common mutation in people of European ancestry, which replaces the amino acid glycine with the amino acid cysteine at position 113 (written as Gly113Cys or G113C). TGM5 gene mutations reduce the amount of transglutaminase 5 that is produced or prevent cells from making any of this enzyme. A shortage of transglutaminase 5 impairs protein cross-linking, which weakens the cornified cell envelope and allows the outermost cells of the epidermis to separate easily from the underlying skin and peel off.
    [Show full text]