All three mammalian MutL complexes are required for repeat expansion in a mouse cell model of the Fragile X-related disorders


Autoři: Carson J. Miller aff001;  Geum-Yi Kim aff001;  Xiaonan Zhao aff001;  Karen Usdin aff001
Působiště autorů: Section on Gene Structure and Disease, Laboratory of Cell and Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America aff001
Vyšlo v časopise: All three mammalian MutL complexes are required for repeat expansion in a mouse cell model of the Fragile X-related disorders. PLoS Genet 16(6): e32767. doi:10.1371/journal.pgen.1008902
Kategorie: Research Article
doi: 10.1371/journal.pgen.1008902

Souhrn

Expansion of a CGG-repeat tract in the 5’ untranslated region of the FMR1 gene causes the fragile X-related disorders (FXDs; aka the FMR1 disorders). The expansion mechanism is likely shared by the 35+ other diseases resulting from expansion of a disease-specific microsatellite, but many steps in this process are unknown. We have shown previously that expansion is dependent upon functional mismatch repair proteins, including an absolute requirement for MutLγ, one of the three MutL heterodimeric complexes found in mammalian cells. We demonstrate here that both MutLα and MutLβ, the two other MutL complexes present in mammalian cells, are also required for most, if not all, expansions in a mouse embryonic stem cell model of the FXDs. A role for MutLα and MutLβ is consistent with human GWA studies implicating these complexes as modifiers of expansion risk in other Repeat Expansion Diseases. The requirement for all three complexes suggests a novel model in which these complexes co-operate to generate expansions. It also suggests that the PMS1 subunit of MutLβ may be a reasonable therapeutic target in those diseases in which somatic expansion is an important disease modifier.

Klíčová slova:

Genome-wide association studies – Guide RNA – Mouse models – Mutation – Nucleases – Oligonucleotides – Point mutation – Polymerase chain reaction


Zdroje

1. Paulson H. Repeat expansion diseases. Handb Clin Neurol. 2018;147:105–23. Epub 2018/01/13. doi: 10.1016/B978-0-444-63233-3.00009-9 29325606; PubMed Central PMCID: PMC6485936.

2. Lozano R, Rosero CA, Hagerman RJ. Fragile X spectrum disorders. Intractable Rare Dis Res. 2014;3(4):134–46. Epub 2015/01/22. doi: 10.5582/irdr.2014.01022 25606363; PubMed Central PMCID: PMC4298643.

3. Polyzos AA, McMurray CT. Close encounters: Moving along bumps, breaks, and bubbles on expanded trinucleotide tracts. DNA Repair (Amst). 2017;56:144–55. Epub 2017/07/12. doi: 10.1016/j.dnarep.2017.06.017 28690053; PubMed Central PMCID: PMC5558859.

4. McGinty RJ, Mirkin SM. Cis- and Trans-Modifiers of Repeat Expansions: Blending Model Systems with Human Genetics. Trends Genet. 2018;34(6):448–65. Epub 2018/03/24. doi: 10.1016/j.tig.2018.02.005 29567336; PubMed Central PMCID: PMC5959756.

5. Morales F, Vasquez M, Santamaria C, Cuenca P, Corrales E, Monckton DG. A polymorphism in the MSH3 mismatch repair gene is associated with the levels of somatic instability of the expanded CTG repeat in the blood DNA of myotonic dystrophy type 1 patients. DNA Repair (Amst). 2016;40:57–66. Epub 2016/03/20. doi: 10.1016/j.dnarep.2016.01.001 26994442.

6. Moss DJH, Pardinas AF, Langbehn D, Lo K, Leavitt BR, Roos R, et al. Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study. Lancet Neurol. 2017;16(9):701–11. Epub 2017/06/24. doi: 10.1016/S1474-4422(17)30161-8 28642124.

7. Bettencourt C, Hensman-Moss D, Flower M, Wiethoff S, Brice A, Goizet C, et al. DNA repair pathways underlie a common genetic mechanism modulating onset in polyglutamine diseases. Ann Neurol. 2016;79(6):983–90. Epub 2016/04/05. doi: 10.1002/ana.24656 27044000; PubMed Central PMCID: PMC4914895.

8. Genetic Modifiers of Huntington's Disease Consortium. Electronic address ghmhe, Genetic Modifiers of Huntington's Disease C. CAG Repeat Not Polyglutamine Length Determines Timing of Huntington's Disease Onset. Cell. 2019;178(4):887–900 e14. Epub 2019/08/10. doi: 10.1016/j.cell.2019.06.036 31398342; PubMed Central PMCID: PMC6700281.

9. Flower M, Lomeikaite V, Ciosi M, Cumming S, Morales F, Lo K, et al. MSH3 modifies somatic instability and disease severity in Huntington's and myotonic dystrophy type 1. Brain. 2019;142(7):1876–86. Epub 2019/06/20. doi: 10.1093/brain/awz115 31216018; PubMed Central PMCID: PMC6598626.

10. Ciosi M, Maxwell A, Cumming SA, Hensman Moss DJ, Alshammari AM, Flower MD, et al. A genetic association study of glutamine-encoding DNA sequence structures, somatic CAG expansion, and DNA repair gene variants, with Huntington disease clinical outcomes. EBioMedicine. 2019;48:568–80. Epub 2019/10/15. doi: 10.1016/j.ebiom.2019.09.020 31607598; PubMed Central PMCID: PMC6838430.

11. Genetic Modifiers of Huntington's Disease C. Identification of Genetic Factors that Modify Clinical Onset of Huntington's Disease. Cell. 2015;162(3):516–26. Epub 2015/08/02. doi: 10.1016/j.cell.2015.07.003 26232222; PubMed Central PMCID: PMC4524551.

12. Zhao XN, Usdin K. FAN1 protects against repeat expansions in a Fragile X mouse model. DNA Repair (Amst). 2018;69:1–5. Epub 2018/07/11. doi: 10.1016/j.dnarep.2018.07.001 29990673; PubMed Central PMCID: PMC6119480.

13. Zhao X, Zhang Y, Wilkins K, Edelmann W, Usdin K. MutLgamma promotes repeat expansion in a Fragile X mouse model while EXO1 is protective. PLoS Genet. 2018;14(10):e1007719. Epub 2018/10/13. doi: 10.1371/journal.pgen.1007719 30312299; PubMed Central PMCID: PMC6200270.

14. Zhao XN, Lokanga R, Allette K, Gazy I, Wu D, Usdin K. A MutSbeta-Dependent Contribution of MutSalpha to Repeat Expansions in Fragile X Premutation Mice? PLoS Genet. 2016;12(7):e1006190. Epub 2016/07/20. doi: 10.1371/journal.pgen.1006190 27427765; PubMed Central PMCID: PMC4948851.

15. Zhao XN, Usdin K. The transcription-coupled repair protein ERCC6/CSB also protects against repeat expansion in a mouse model of the fragile X premutation. Hum Mutat. 2015;36(4):482–7. Epub 2015/03/03. doi: 10.1002/humu.22777 25726753; PubMed Central PMCID: PMC4382389.

16. Zhao XN, Kumari D, Gupta S, Wu D, Evanitsky M, Yang W, et al. Mutsbeta generates both expansions and contractions in a mouse model of the Fragile X-associated disorders. Hum Mol Genet. 2015;24(24):7087–96. Epub 2015/10/01. doi: 10.1093/hmg/ddv408 26420841; PubMed Central PMCID: PMC4654059.

17. Zhao XN, Usdin K. Gender and cell-type-specific effects of the transcription-coupled repair protein, ERCC6/CSB, on repeat expansion in a mouse model of the fragile X-related disorders. Hum Mutat. 2014;35(3):341–9. Epub 2013/12/20. doi: 10.1002/humu.22495 24352881; PubMed Central PMCID: PMC4067466.

18. Lokanga RA, Zhao XN, Usdin K. The mismatch repair protein MSH2 is rate limiting for repeat expansion in a fragile X premutation mouse model. Hum Mutat. 2014;35(1):129–36. Epub 2013/10/17. doi: 10.1002/humu.22464 24130133; PubMed Central PMCID: PMC3951054.

19. Tome S, Manley K, Simard JP, Clark GW, Slean MM, Swami M, et al. MSH3 polymorphisms and protein levels affect CAG repeat instability in Huntington's disease mice. PLoS Genet. 2013;9(2):e1003280. Epub 2013/03/08. doi: 10.1371/journal.pgen.1003280 23468640; PubMed Central PMCID: PMC3585117.

20. Halabi A, Ditch S, Wang J, Grabczyk E. DNA mismatch repair complex MutSbeta promotes GAA.TTC repeat expansion in human cells. J Biol Chem. 2012;287(35):29958–67. Epub 2012/07/13. doi: 10.1074/jbc.M112.356758 22787155; PubMed Central PMCID: PMC3436174.

21. Gannon AM, Frizzell A, Healy E, Lahue RS. MutSbeta and histone deacetylase complexes promote expansions of trinucleotide repeats in human cells. Nucleic Acids Res. 2012;40(20):10324–33. Epub 2012/09/04. doi: 10.1093/nar/gks810 22941650; PubMed Central PMCID: PMC3488247.

22. Foiry L, Dong L, Savouret C, Hubert L, te Riele H, Junien C, et al. Msh3 is a limiting factor in the formation of intergenerational CTG expansions in DM1 transgenic mice. Hum Genet. 2006;119(5):520–6. Epub 2006/03/23. doi: 10.1007/s00439-006-0164-7 16552576.

23. van den Broek WJ, Nelen MR, Wansink DG, Coerwinkel MM, te Riele H, Groenen PJ, et al. Somatic expansion behaviour of the (CTG)n repeat in myotonic dystrophy knock-in mice is differentially affected by Msh3 and Msh6 mismatch-repair proteins. Hum Mol Genet. 2002;11(2):191–8. Epub 2002/01/26. doi: 10.1093/hmg/11.2.191 11809728.

24. Halabi A, Fuselier KTB, Grabczyk E. GAA*TTC repeat expansion in human cells is mediated by mismatch repair complex MutLgamma and depends upon the endonuclease domain in MLH3 isoform one. Nucleic Acids Res. 2018;46(8):4022–32. Epub 2018/03/13. doi: 10.1093/nar/gky143 29529236; PubMed Central PMCID: PMC5934671.

25. Pinto RM, Dragileva E, Kirby A, Lloret A, Lopez E, St Claire J, et al. Mismatch repair genes Mlh1 and Mlh3 modify CAG instability in Huntington's disease mice: genome-wide and candidate approaches. PLoS Genet. 2013;9(10):e1003930. Epub 2013/11/10. doi: 10.1371/journal.pgen.1003930 24204323; PubMed Central PMCID: PMC3814320.

26. Cannavo E, Marra G, Sabates-Bellver J, Menigatti M, Lipkin SM, Fischer F, et al. Expression of the MutL homologue hMLH3 in human cells and its role in DNA mismatch repair. Cancer Res. 2005;65(23):10759–66. Epub 2005/12/03. doi: 10.1158/0008-5472.CAN-05-2528 16322221.

27. Kulak NA, Pichler G, Paron I, Nagaraj N, Mann M. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat Methods. 2014;11(3):319–24. Epub 2014/02/04. doi: 10.1038/nmeth.2834 24487582.

28. Raschle M, Marra G, Nystrom-Lahti M, Schar P, Jiricny J. Identification of hMutLbeta, a heterodimer of hMLH1 and hPMS1. J Biol Chem. 1999;274(45):32368–75. Epub 1999/11/05. doi: 10.1074/jbc.274.45.32368 10542278.

29. Genetic Modifiers of Huntington's Disease Consortium. CAG Repeat Not Polyglutamine Length Determines Timing of Huntington's Disease Onset. Cell. 2019;178(4):887–900 e14. Epub 2019/08/10. doi: 10.1016/j.cell.2019.06.036 31398342; PubMed Central PMCID: PMC6700281.

30. Gomes-Pereira M, Fortune MT, Ingram L, McAbney JP, Monckton DG. Pms2 is a genetic enhancer of trinucleotide CAG.CTG repeat somatic mosaicism: implications for the mechanism of triplet repeat expansion. Hum Mol Genet. 2004;13(16):1815–25. Epub 2004/06/17. doi: 10.1093/hmg/ddh186 15198993.

31. Bourn RL, De Biase I, Pinto RM, Sandi C, Al-Mahdawi S, Pook MA, et al. Pms2 suppresses large expansions of the (GAA.TTC)n sequence in neuronal tissues. PLoS One. 2012;7(10):e47085. Epub 2012/10/17. doi: 10.1371/journal.pone.0047085 23071719; PubMed Central PMCID: PMC3469490.

32. Kadyrov FA, Dzantiev L, Constantin N, Modrich P. Endonucleolytic function of MutLalpha in human mismatch repair. Cell. 2006;126(2):297–308. Epub 2006/07/29. doi: 10.1016/j.cell.2006.05.039 16873062.

33. Gazy I, Miller C, Kim G-Y, Usdin K. CGG repeat expansion, and elevated Fmr1 transcription and mitochondrial copy number in a new Fragile X PM mouse embryonic stem cell model. Frontiers in Cell and Devel Biol. In press. doi: 10.3389/fcell.2020.00482

34. Du J, Campau E, Soragni E, Ku S, Puckett JW, Dervan PB, et al. Role of mismatch repair enzymes in GAA.TTC triplet-repeat expansion in Friedreich ataxia induced pluripotent stem cells. J Biol Chem. 2012;287(35):29861–72. Epub 2012/07/17. doi: 10.1074/jbc.M112.391961 22798143; PubMed Central PMCID: PMC3436184.

35. Du J, Campau E, Soragni E, Jespersen C, Gottesfeld JM. Length-dependent CTG.CAG triplet-repeat expansion in myotonic dystrophy patient-derived induced pluripotent stem cells. Hum Mol Genet. 2013;22(25):5276–87. Epub 2013/08/13. doi: 10.1093/hmg/ddt386 23933738; PubMed Central PMCID: PMC3842182.

36. Zhao X, Gazy I, Hayward B, Pintado E, Hwang YH, Tassone F, et al. Repeat Instability in the Fragile X-Related Disorders: Lessons from a Mouse Model. Brain Sci. 2019;9(3):E52. Epub 2019/03/06. doi: 10.3390/brainsci9030052 30832215; PubMed Central PMCID: PMC6468611.

37. Mollersen L, Rowe AD, Larsen E, Rognes T, Klungland A. Continuous and periodic expansion of CAG repeats in Huntington's disease R6/1 mice. PLoS Genet. 2010;6(12):e1001242. Epub 2010/12/21. doi: 10.1371/journal.pgen.1001242 21170307; PubMed Central PMCID: PMC3000365.

38. Toledo M, Sun X, Brieno-Enriquez MA, Raghavan V, Gray S, Pea J, et al. A mutation in the endonuclease domain of mouse MLH3 reveals novel roles for MutLgamma during crossover formation in meiotic prophase I. PLoS Genet. 2019;15(6):e1008177. Epub 2019/06/07. doi: 10.1371/journal.pgen.1008177 31170160; PubMed Central PMCID: PMC6588253.

39. Fischer JM, Dudley S, Miller AJ, Liskay RM. An intact Pms2 ATPase domain is not essential for male fertility. DNA Repair (Amst). 2016;39:46–51. Epub 2016/01/13. doi: 10.1016/j.dnarep.2015.12.011 26753533; PubMed Central PMCID: PMC4766077.

40. Johnson JR, Erdeniz N, Nguyen M, Dudley S, Liskay RM. Conservation of functional asymmetry in the mammalian MutLalpha ATPase. DNA Repair (Amst). 2010;9(11):1209–13. Epub 2010/09/25. doi: 10.1016/j.dnarep.2010.08.006 20864418; PubMed Central PMCID: PMC2970632.

41. Drummond JT, Anthoney A, Brown R, Modrich P. Cisplatin and adriamycin resistance are associated with MutLalpha and mismatch repair deficiency in an ovarian tumor cell line. J Biol Chem. 1996;271(33):19645–8. Epub 1996/08/16. doi: 10.1074/jbc.271.33.19645 8702663.

42. Prolla TA, Baker SM, Harris AC, Tsao JL, Yao X, Bronner CE, et al. Tumour susceptibility and spontaneous mutation in mice deficient in Mlh1, Pms1 and Pms2 DNA mismatch repair. Nat Genet. 1998;18(3):276–9. Epub 1998/03/21. doi: 10.1038/ng0398-276 9500552.

43. Hensman Moss DJ, Pardinas AF, Langbehn D, Lo K, Leavitt BR, Roos R, et al. Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study. Lancet Neurol. 2017;16(9):701–11. Epub 2017/06/24. doi: 10.1016/S1474-4422(17)30161-8 28642124.

44. Ranjha L, Anand R, Cejka P. The Saccharomyces cerevisiae Mlh1-Mlh3 heterodimer is an endonuclease that preferentially binds to Holliday junctions. J Biol Chem. 2014;289(9):5674–86. Epub 2014/01/21. doi: 10.1074/jbc.M113.533810 24443562; PubMed Central PMCID: PMC3937642.

45. Kadyrova LY, Gujar V, Burdett V, Modrich P, Kadyrov FA. Human MutLγ, the MLH1-MLH3 heterodimer, has a novel endonuclease activity that promotes DNA expansion. Proc Natl Acad Sci USA. 2020.

46. Pluciennik A, Burdett V, Baitinger C, Iyer RR, Shi K, Modrich P. Extrahelical (CAG)/(CTG) triplet repeat elements support proliferating cell nuclear antigen loading and MutLalpha endonuclease activation. Proc Natl Acad Sci U S A. 2013;110(30):12277–82. Epub 2013/07/11. doi: 10.1073/pnas.1311325110 23840062; PubMed Central PMCID: PMC3725108.

47. van Oers JM, Roa S, Werling U, Liu Y, Genschel J, Hou H Jr., et al. PMS2 endonuclease activity has distinct biological functions and is essential for genome maintenance. Proc Natl Acad Sci U S A. 2010;107(30):13384–9. Epub 2010/07/14. doi: 10.1073/pnas.1008589107 20624957; PubMed Central PMCID: PMC2922181.

48. Duroc Y, Kumar R, Ranjha L, Adam C, Guerois R, Md Muntaz K, et al. Concerted action of the MutLbeta heterodimer and Mer3 helicase regulates the global extent of meiotic gene conversion. Elife. 2017;6. Epub 2017/01/05. doi: 10.7554/eLife.21900 28051769; PubMed Central PMCID: PMC5215242.

49. Hayward B, Steinbach PJ, Usdin K. A point mutation in the nuclease domain of MLH3 eliminates repeat expansions in a mouse stem cell model of the Fragile X-related disorders. Nucleic Acids Res. In revision.

50. Manhart CM, Ni X, White MA, Ortega J, Surtees JA, Alani E. The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans. PLoS Biol. 2017;15(4):e2001164. Epub 2017/04/30. doi: 10.1371/journal.pbio.2001164 28453523; PubMed Central PMCID: PMC5409509.

51. Cannavo E, Sanchez A, Anand R, Ranjha L, Hugener J, Adam C, et al. Regulation of the MLH1-MLH3 endonuclease in meiosis. bioRxiv. 2020:2020.02.12.946293. doi: 10.1101/2020.02.12.946293

52. Adam R, Spier I, Zhao B, Kloth M, Marquez J, Hinrichsen I, et al. Exome Sequencing Identifies Biallelic MSH3 Germline Mutations as a Recessive Subtype of Colorectal Adenomatous Polyposis. Am J Hum Genet. 2016;99(2):337–51. Epub 2016/08/02. doi: 10.1016/j.ajhg.2016.06.015 27476653; PubMed Central PMCID: PMC4974087.

53. Yin J, Kong D, Wang S, Zou TT, Souza RF, Smolinski KN, et al. Mutation of hMSH3 and hMSH6 mismatch repair genes in genetically unstable human colorectal and gastric carcinomas. Hum Mutat. 1997;10(6):474–8. Epub 1997/01/01. doi: 10.1002/(SICI)1098-1004(1997)10:6<474::AID-HUMU9>3.0.CO;2-D 9401011.

54. Marra G, Iaccarino I, Lettieri T, Roscilli G, Delmastro P, Jiricny J. Mismatch repair deficiency associated with overexpression of the MSH3 gene. Proc Natl Acad Sci U S A. 1998;95(15):8568–73. Epub 1998/07/22. doi: 10.1073/pnas.95.15.8568 9671718; PubMed Central PMCID: PMC21116.

55. Lipkin SM, Wang V, Stoler DL, Anderson GR, Kirsch I, Hadley D, et al. Germline and somatic mutation analyses in the DNA mismatch repair gene MLH3: Evidence for somatic mutation in colorectal cancers. Hum Mutat. 2001;17(5):389–96. Epub 2001/04/24. doi: 10.1002/humu.1114 11317354.

56. Taylor NP, Powell MA, Gibb RK, Rader JS, Huettner PC, Thibodeau SN, et al. MLH3 mutation in endometrial cancer. Cancer Res. 2006;66(15):7502–8. Epub 2006/08/04. doi: 10.1158/0008-5472.CAN-06-0248 16885347.

57. Liu HX, Li Y, Jiang XD, Yin HN, Zhang L, Wang Y, et al. Mutation screening of mismatch repair gene Mlh3 in familial esophageal cancer. World J Gastroenterol. 2006;12(33):5281–6. Epub 2006/09/19. doi: 10.3748/wjg.v12.i33.5281 16981255; PubMed Central PMCID: PMC4088192.

58. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281–308. Epub 2013/10/26. doi: 10.1038/nprot.2013.143 24157548; PubMed Central PMCID: PMC3969860.

59. Heigwer F, Kerr G, Boutros M. E-CRISP: fast CRISPR target site identification. Nat Methods. 2014;11(2):122–3. Epub 2014/02/01. doi: 10.1038/nmeth.2812 24481216.

60. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013;31(9):827–32. Epub 2013/07/23. doi: 10.1038/nbt.2647 23873081; PubMed Central PMCID: PMC3969858.

61. Veres A, Gosis BS, Ding Q, Collins R, Ragavendran A, Brand H, et al. Low incidence of off-target mutations in individual CRISPR-Cas9 and TALEN targeted human stem cell clones detected by whole-genome sequencing. Cell Stem Cell. 2014;15(1):27–30. Epub 2014/07/06. doi: 10.1016/j.stem.2014.04.020 24996167; PubMed Central PMCID: PMC4082799.

62. Kim D, Luk K, Wolfe SA, Kim JS. Evaluating and Enhancing Target Specificity of Gene-Editing Nucleases and Deaminases. Annu Rev Biochem. 2019;88:191–220. Epub 2019/03/19. doi: 10.1146/annurev-biochem-013118-111730 30883196.

63. Meier JA, Zhang F, Sanjana NE. GUIDES: sgRNA design for loss-of-function screens. Nat Methods. 2017;14(9):831–2. Epub 2017/09/01. doi: 10.1038/nmeth.4423 28858339; PubMed Central PMCID: PMC5870754.

64. Lee JM, Zhang J, Su AI, Walker JR, Wiltshire T, Kang K, et al. A novel approach to investigate tissue-specific trinucleotide repeat instability. BMC Syst Biol. 2010;4:29. Epub 2010/03/23. doi: 10.1186/1752-0509-4-29 20302627; PubMed Central PMCID: PMC2856555.

65. Zhao X, Lu H, Dagur PK, Usdin K. Isolation and Analysis of the CGG-Repeat Size in Male and Female Gametes from a Fragile X Mouse Model. Methods Mol Biol. 2020;2056:173–86. Epub 2019/10/06. doi: 10.1007/978-1-4939-9784-8_11 31586348.

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Genetika Reprodukční medicína

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PLOS Genetics


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