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Introduction Center for Medical Genetics

Introduction Center for Medical Genetics. Staff (81) Clinicians and psychologists Laboratory supervisors Researchers Laboratory technicians Secretary. Introduction Center for Medical Genetics. Diagnosis of genetic disorders Clinical assesment Laboratory investigations (three labs)

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Introduction Center for Medical Genetics

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  1. Introduction Center for Medical Genetics Staff (81) Clinicians and psychologists Laboratory supervisors Researchers Laboratory technicians Secretary

  2. Introduction Center for Medical Genetics Diagnosis of genetic disorders Clinical assesment Laboratory investigations (three labs) Counseling

  3. Cursus Human Molecular Genetics Les 1 : From human cytogenetics to molecular cytogenetics Les 2 : Monogenic disorders Les 3 : Familial cancer Les 4 : Multifactorial genetic disorders Les 5 : Diagnosis and Research in Human Genetics

  4. From human cytogenetics to molecular cytogenetics • introduction • historical overview (the birth of human • cytogenetics) • progress in (molecular) cytogenetics • general aspects of (molecular) cytogenetics • molecular mechanisms for constitutional • chromosomal rearrangements in humans

  5. The birth of human cytogenetics • 1956: Tjio and Levan count the full complement • of 46 human chromosomes

  6. The birth of human cytogenetics • 1956: Tjio and Levan count the full complement • of 46 human chromosomes • serendipitous addition of water to a suspension • of fixed cells • 3 years after description of DNA structure • 30 years after count of 48 chromosomes by • Thomas Painter

  7. The birth of human cytogenetics • human chromosomes have a morphology which • allows classification

  8. The birth of human cytogenetics • rapidly associations were found between • human diseases (syndromes) and specific • chromosome abnormalities • 1959 • Lejeune et al : +21 in Down syndrome • Ford et al. : 45,X in Turner syndrome • Jacobs et al : 47,XXY in Klinefelter syndrome • 1960 Nowel and Hungerford • Philadelphia chromosome in CML • 1973 Rowley: t(9;22)(q34;q11) in CML

  9. The birth of human cytogenetics rapidly associations were found between human diseases (syndromes) and specific chromosome abnormalities 1963 chromosome 5 short arm partial deletion in Cri du Chat syndrome 1963 D-chromosome deletion in patient with bilateral retinoblastoma

  10. cordocentesis amniocentesis Chorion villi sampling Preimplantation genetic diagnosis Prenatal diagnosis www.visembryo.com/baby/hp.html

  11. Further progress in human cytogenetics is fueled by technical innovations (I) • 1968 Caspersson et al • differential staining of chromosomes produces • a recognizable banding pattern (chromosomal • barcode) along the length of the chromosomes • chromosome bands are related to differences in • base pair composition, gene density, repetitive • elements, chromatin packaging but molecular • basis is not understood • greatly facilitates classification and recognition • of structural aberations

  12. general aspects of (molecular) cytogenetics

  13. ISCN 1995 International System for Human Cytogenetic Nomenclature groep A (1-3) groep B (4-5) groep C (6-12, X) groep D (13-15) groep E (16-18) groep F (19-20)

  14. general aspects of (molecular) cytogenetics

  15. general aspects of (molecular) cytogenetics chromosomal rearrangements • numerical chromosome changes/aneuploidy • result from errors occurring during • meiotic or mitotic segregation • structural chromosome changes • translocations • inversions • insertions • deletions • duplications

  16. CYTOGENETICA EN MOLECULAIRE CYTOGENETICA: CONSTITUTIONELE EN VERWORVEN CHROMOSOMALE DEFECTEN

  17. general aspects of (molecular) cytogenetics

  18. general aspects of (molecular) cytogenetics

  19. general aspects of (molecular) cytogenetics

  20. general aspects of (molecular) cytogenetics reciprocal translocation

  21. ISCN 1995 International System for Human Cytogenetic Nomenclature http://www.waisman.wisc.edu/cytogenetics/abnormalities/abnormalities.html Robertsonian translocation 45,XX,der(13;14)(q10;q10) Reciprocal translocation 46,XY,t(6;9)(q24;p23)

  22. ISCN 1995 International System for Human Cytogenetic Nomenclature http://www.waisman.wisc.edu/cytogenetics/abnormalities/abnormalities.html Reciprocal translocation (unbalanced) 46,XY,t(6;9)(q24;p23) 46,XY,der(6)t(6;9)(q24;p23)

  23. ISCN 1995 International System for Human Cytogenetic Nomenclature insertion inversion 46,XY,ins(5;2)(p14;q22q32) 46,XX,inv(9)(p13q13)

  24. ISCN 1995 International System for Human Cytogenetic Nomenclature duplication deletion del(18)(pterp11.2) del(18)(p11.2) 46,X,dup(X)(p11.2p22.1)

  25. Further progress in human cytogenetics is fueled by technical innovations (II) methods for mapping (disease) genes based upon chromosomal rearrangements • Somatic cell hybrids • flow sorted chromosomes • FISH

  26. Further progress in human cytogenetics is fueled by technical innovations (II) Somatic cell hybrids

  27. Further progress in human cytogenetics is fueled by technical innovations (II)

  28. Molecular mechanisms for constitutional chromosomal rearrangements in humans Positional cloning of t(1;17) breakpoints • constitutional (1;17)(p36.2;q11.2) in patient • with neuroblastoma • 1p36 region is frequently lost in NB • association of a translocation with a particular disease • phenotype may point at the chromosomal localisation • of the disease gene • additional evidence from eg LOH, linkage,mouse,… • positional cloning: cloning of disease gene based upon • the assumption of the chromosomal localisation • physical mapping, identification of candidate genes • mutation analysis, expression studies, functional evidence

  29. Molecular mechanisms for constitutional chromosomal rearrangements in humans Positional cloning of t(1;17) breakpoints

  30. Further progress in human cytogenetics is fueled by technical innovations • late ’80ies introduction of FISH • significant increase of sensitivity • (10.000x) • new possiblities eg interphase • various applications eg gene mapping, • genetic diagnosis, research • “the FISH have spawned” • CGH • M-FISH/SKY • FICTION • fibre FISH

  31. Fluorescence in situ hybridisation DNA Commercial Cot1 DNA Labeling: nick translation Mix 10ml culture Denaturation and incubation at 37°C Wash, detection and counterstain Denaturation Hybridisatie o/n

  32. 2 X chromosome 13 2 X chromosome 18 2 X chromosome 21 1 X chromosome Y Control lymphocytes (FISH 952-35)

  33. part I CGH

  34. part I CGH

  35. part I disadvantages limited resolution (~10 Mb del/dup) laborious only gains and losses / no balanced rearrangements no information on the nature of the aberrations CGH advantages whole genome in 1 experiment no need to culture tumor cells sensitive detection of gene amplification retrospective analysis

  36. Molecular mechanisms for constitutional chromosomal rearrangements in humans • chromosomal rearrangements require the formation • of double strand breaks (DSBs) and subsequent • rejoining of the broken ends between two (or more) • breakpoints • exogenous causes of structural aberrations • X-rays, -rays, -particles and other forms of • ionizing radiation • cause formation of oxidants which are powerful • clastogens • duration of exposure

  37. Molecular mechanisms for constitutional chromosomal rearrangements in humans • exogenous causes of structural aberrations • chemicals: alkylating agents, purine and pyrimidine • analoges, alkyl epoxides, aromatic amines, nitroso • compounds and heavy metals • most often generation of breaks at G2 • viral infections • lesions may undergo repair or misrepair by a wide range • of DNA repair systems

  38. Molecular mechanisms for constitutional chromosomal rearrangements in humans • endogenous causes of structural aberrations • rare autosomal recessive chromosome breakage syndromes • caused by defective DNA repair enzymes • (AT, ATM; BS, BLM; NBS, NBS1) • transposable elements • short and long interspersed elements (LINE, SINE) • 300 bp Alu (every 4 kb, gene rich), longer LINE (gene poor) • segmental duplication, gene duplication • fragile sites

  39. Molecular mechanisms for constitutional chromosomal rearrangements in humans • Segmental duplications: an ‘expanding’ role in genomic • instability and disease. Emanuel and Shaikh, Nature Reviews • Genetics, Volume 2, October 2001, 791-800. • Segmental duplications = region or chromosome specific • low-copy repeats, new class of repetitive DNA elements • recently identified • resulting genetic aberrations • deletions • interstitial duplications • translocations • inversions • marker chromosomes

  40. Molecular mechanisms for constitutional chromosomal rearrangements in humans DiGeorge/velo-cardio-facial syndrome recurrent reciprocal translocation t(11;22) cat eye syndrome (CES)

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