Diapozitivul 1

Human chromosomes2Human chromosomes34Origin of gonosomes

56 Chromosomal number and structural abnormalities induce developmental abnormalities - sundromes7The shape of chromosome depends on the position of centromere

Structure of metaphase chromosomes. Rchromosomal landmarksSecondary constrictionSatellite CentromerePrimary constrictionSister chromatids8

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Human karyotype

10Classification of chromosomesBy length:LargeMediumSmallBy shape:MetacentricSubmetacentricAcrocentricBy type:AutosomesGonosomesBy presence of other landmarks:h on p armh on q armsatellitesGroups:A 1-3B 4,5C X, 6-12D 13-15E 16-18F 19,20G 21, 22, Y

11Human karyotype and chromosomal formula12Human karyotype and chromosomal formulaMethods of chromosome analysis

He is 4 years old (looks like 2 y.o.)Knows few wordsInadequate reactionAbnormal faceShort handsHeart abnormalities, kidney abnormalities

Plurimapformative syndromeWhat is his karyotype?14

1. Collecting of nuclear cells (just leucocytes if blood is used)152. Cell culturing cells in different stages of cell cycleThermostat 37 C72 hoursBlood cells+ growth broth Cells in different stages of cell cycleProliferationAfter 72 hours16

173. Preparation of chromosomes for analysisCells in different stages of cell cycleHypotonic solutionCytostatic - colchicine Mitosis arrestingCell sedimentationChromosome dispersing 18Slide with fixed cellsSlide with metaphase platesPainting4. Painting of chromosomes

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205. Karyotype analysis

Printing of pictureCutting individual chromosomesPreparation of karyotype21

47,XY,+21 Down syndrom22Cytogenetic methods used for karyotype analysis:Analysis of human karyotypeMetaphase chromosomesPrometapfase chromosomesInterphase chromosomesUniform paintingG,Q,R,C,T bandingG, R banding X and Y sex chromatin analysisMolecular citogenetical methodsFISH, mFISHSKYCGH*** No of bands per haploid set: 300-400 m / 550 pm / 850p2425

Type of bandingUsed dyeGGiemsa QQuinacrin(fluorescent) R (revers) Giemsa or fluorescentC (centromere) Giemsa or fluorescentT (telomere) Giemsa or fluorescentChromosome stainingHomogenousBanding

G

Q

R

C

TBands G+ = Q+ = R Bands G- = Q- = R +

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How the same chromosome is painted in different patterns?27

45,X

47,XX,+18

69,XXX

47,XXY

46,XX,5p-

47,XX,+13

47,XX,+21

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37Uniform staining

38C banding

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40Chromosomal bandingChromosomeBandArmSubband4p22.25q13.49p21.3Region

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46,XY,del(1)(p11-p34)1p351p221q231q414246,XY,del(1)(p11-p34)

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46,Y,del(X)(q12.1-q24.3)4445

Chromosome 4Identification of X and 18Identification of X, Y and 18Identification of 18 of 21Identification of 18 and 21FISH in chromosomal analysis

46,XX47,XX,+21

47,XX,+2147,XX,+2146

47SKY

48mFISH

49

50Karyotype46,XX46,XY46,XX,9qh+46,XY,16qh-46,XX,14s++47,XX,+2145,XY,-1248,XXXY45,X69,XXX69,XXY46,XX,1q-46,XY,16p+46,X,r(X)46,XY,del(5p)46,XX,t(12,22)NormalNormal polymorphismsAneuploidyPolyploidyChromosomal abberations51Abnormal phenotype

invtrobBalanced structural aberrations

Fenotip patologicTrisomy 47,XXX; 47,XXY; 47,+21; 47,+13; 47,+18; 47,+8Monosomy 45,XAneuploidiesAbnormal phenotype

Chrs D and G variations in ph or s:Chrs 1,9,16,9,Y variations in qhPolymorphismsNormal phenotype46,XX46,XYNormal karyotypeFenotip patologicdeldupriUnbalanced structural aberrations

52??? Polymorphismsp and q contain coding and non-coding regionsp+ or p- - abnormality q+ or q- - abnormalityc longer or shorter polymorphismt longer or shorter polymorphismh+ or h- - polymorphisms+ or s- - polymorphism53Sex chromatin analysisX chromatin analysis = Barr body test identification of non-active X, in interphase nuclei of somatic cellsY chromatin analysis = F body test identification of 2/3 of qY in interphase nuclei of somatic cells or sperms54Normal karyotype

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Y chromosome - 397 genes: regulatory masculinisation genes pseudogenes !!! q arm contains constitutive heterochromatinX chromosome - 1606 genes: Structural somatic genesRegulatory feminisation genes;Structural feminisation genes; Structural masculinisation genes

56 !!! Important57How to explain this?58

45,X47,XXY59

46,XX

46,XYActive X - euchromatinActive X - euchromatinY chromosome p arm active q arm - inactiveInactive X - heterochromatin

F body = 0,25 m

Barr body = 1m60

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62Barr body X sex chromatinNrBarr = X - 1

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Barr body testXXXXXXXXXXXXXYYYF body test48,XXXY45,X50,XXXXYY49,XXXXX64Barr body testX sexual chromatin: represents an inactive X, facultative heterochromatin, in somatic cells 46,XX; represents a Barr body 1m in diameter;Barr body test is useful for identification of No of X chromosomes in somatic cells: No X = No Barr + 1.46,XX 1 Barr body46,XY 0 47,XXX 247,XXY - 145,X 048,XXXX 3!!! M. Lyon (1961) - explanation of Barr body65

Mary Lyon hypothesis

After 16th day!!! In 46,XX cells after 16th day of development only one X remains activeIn 50% of cells maternal X is active, in 50% - paternal X66

46,XX45,X active +X paternal inactive45,X active + X maternal inactive67F body testY sexual chromatin: represents 2/3 of q arm of Y chromosome, its a constitutive heterochromatin in somatic cells or 50% of sperms (23,Y only); represents fluorescent F body 0,25m in diameter; F body test is useful for prenatal identification of gender: No Y = No F bodies46,XX 046,XY 1 47,XYY 247,XXY - 148,XXYY 246,X,i(Yp) 046,X,i(Yq) 1 (0,5m)68