cytogenetics eng final [kompatibilitási mód] · the goal of cytogenetics: 1. diagnosis of...

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Cytogenetics

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Cytogenetics is the study of chromosomes and their role in heredity.

The goal of cytogenetics:

1. diagnosis of chromosomal abnormalities.

2. localisation of any (often abnormal) chromosomal region/DNA sequence.

Cytogenetics




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Chromatin: non condensed DNA with proteins attached (interphase of the cell cycle)

Chromosome: condensed DNA with proteins attached (M phase of the cell cycle)

Human cells contain 46 chromosome, 44 autosome and 2 sex chromosome.

A karyotype is the number and appearance of chromosomes in the nucleus of a eukaryotic cell. The term is also used for the complete set of chromosomes in a species, or an individual organism.

The chromosomes are depicted (by rearranging a microphotograph) in a standard format known as a karyogram or idiogram: in pairs, ordered by size and position of centromere for chromosomes of the same size.

Basic Cytogenetic Terms




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Human chromosomes are examined in dividing cells (bone marrow/placental cells, lymphocytes).

The cell cycle and the detection of the chromosomes




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1. chr. 1. chr. (human)(human)

DNA length 50 mm

chr. length 3-4 µm

10.000 x 10.000 x condensationcondensation

The steps of chromosome assembly




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The steps of chromosome assembly




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The history of human chromosome identification

1879. Arnold: First visualization of human chromosomes.

1888. Waldeyer: The word chromosome (chroma: color, soma: body)

1882. Walther Flemming: 20-28 chromosomes in cells of cornea

1921. T.S. Painter: 48 human chromosomes, X & Y chromosomes (Science)

1956. Jo Hin Tijo és Albert Levan : 46 human chromosomes (Hereditas)

1959. Lejeune: trisomy 21=Down syndrome




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1960. Denver: chromosomes s numbered ((1-22) based on their size

1963. London: chromosome grouping (A-G)

1966. Chicago: big chromosome syndromes

1971. Paris, 1976. Mexico, 1978. Stockholm: chromosome banding

1995. ISCN : International System for Human Cytogenetic Nomenclature

Kariotyping conferences

The history of human chromosome identification




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46, XX




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46, XY




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Chromosome structure




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Chromosome bandingThe chromosome banding techniques use different chemicals The chromosome banding techniques use different chemicals

to stain the chromosomes in order to identify them or to show to stain the chromosomes in order to identify them or to show

chromosomal rearrangements. Based on the stains used the chromosomal rearrangements. Based on the stains used the

following banding techniques exist:following banding techniques exist:

•• GG--banding:banding: Giemsa staining

•• RR--banding:banding: (reverse) modified Giemsa staining

•• CC--banding:banding: centromer specific staining

•• TT--banding:banding: telomer specific staining

•• QQ--bandingbanding: : quinacrin staining (fluorescent)Starting from the centromere, the short and the long chromosome arms are numbered based on the banding pattern.




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QQ--bandingbandingG-banding

R-banding C-banding T-banding

Chromosome banding




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Human diseases resulting from altered kariotype are mostly caused by ANEUPLOIDIES, TRANSLOCATIONS, and DELETIONS.

Chromosomal abnormalities




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ANEUPLOIDY

ANEUPLOIDY is the abnormal number of chromosomes. The most frequent aneuploidia is trisomy, having three instead of two chromosomes.




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MITOTIC NON-DISJUNCTION

MEIOTICNON-DISJUNCTION




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MEIOTIC NON-DISJUNCTION—> ANEUPLOIDY




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MEIOTIC/MITOTIC NON-DISJUNCTION




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Chromosome number abnormalitiesEuploid chromosome mutation: Triploidity,Triploidity, PoliploidPoliploidityityAneuploid chromosome mutationAutosomal occurence Sex chromosomal occurence

Trisomy21. trisomyDown-syndrome

1/700 47, XXX Triplo-X-syndrome

1/1000 female

18. trisomyEdwards- syndrome

1/13000 47, XXY Klinefelter-syndrome

1/1000 male

13. trisomyPatau-syndrome

1/15000 47, XYY Dupla-Y-syndrome

1/1000 male

Rare trisomies 3-,7-,8-,9-,12-,14-,15-,19-,22-esMonosomy 45, X0 Turner-

syndrome1/2500 female




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AUTOSOMAL ANEUPLOIDIES: DOWN SYNDROME




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AUTOSOMAL ANEUPLOIDIES: DOWN SYNDROME

trisomy of the 21. chromosome




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MATERNAL AGE & THE DOWN SYNDROME




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trisomy of the 18. chromosome

AUTOSOMAL ANEUPLOIDIES: EDWARDS SYNDROME




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SEX CHROMOSOMAL ANEUPLOIDIES:




25Karyotype: 45X0

SEX CHROMOSOMAL ANEUPLOIDIES:TURNER SYNDROME




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SEX CHROMOSOMAL ANEUPLOIDIES:KLINEFELTER SYNDROME

Karyotype: 47XXY




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SEX CHROMOSOMAL ANEUPLOIDIES:TRIPLE X SYNDROME

Karyotype: 47XXX




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SEX CHROMOSOMAL ANEUPLOIDIES:YY SYNDROME

Karyotype: 47XYY




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CLASSIFICATION OF STRUCTURAL CHROMOSOME ABNORMALITIES BASED ON THE NUMBER OF

CHROMOSOMAL BREAK POINTS

terminális deléció

1 törés

reciprok transzlokáció centrikus fúzióvagy

Robertson-féletranszlokáció

2 különböző kromoszómán

paracentrikus inverzió

uazon a kromoszómakaron

gyűrű kromoszóma pericentrikus inverzió

ellentétes kromoszómakaron

uazon a kromoszómán

2 törés

inszerció

3 törés2 különböző

kromoszómán

szerkezeti kromoszóma aberráció

3 breaks3 breaks2 breaks2 breaks1 break1 breakOn 2 different On 2 different

chromosomeschromosomes

Structural abberation of chromosomesStructural abberation of chromosomes

Terminal deletionTerminal deletion On 2 different chromosomesOn 2 different chromosomes InsertionInsertionOn same chromosomeOn same chromosome

Reciprocal Reciprocal translocationtranslocation

On same chromosome On same chromosome armarm

Central fusionCentral fusion

or or

Robertson Robertson translocationtranslocation

On opposite On opposite chromosome armchromosome arm

Paracentric Paracentric inversioninversion

Ring Ring chromosomechromosome

Pericentric Pericentric inversioninversion




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DELETION

A piece of the chromosome is lost

Deletion mapping

Intersticial deletion - Prader-Willi; Angelman syndromes del 5(q11-13)Williams syndrome

Terminal deletion - telomer is lost too – severe symptomsCri du chat syndrome




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TRANSLOCATION

Reciprocal translocation:Reciprocal translocation: breakpoints are on 2 homologous or on 2 different chromosomes

break points are mostly in non coding regions

balanced translocation

breakpoints inside genesbreakpoints inside genes - genes with altered function - genes with altered activity- genes with altered expression- genes with loss of function




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RING CHROMOSOME

forms after tforms after telomere deletion




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INVERSION

Paracentric inversion Pericentrikus inversion




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INHERITED DELETIONSINHERITED DELETIONS

DELETIONDELETION SYMPTOMSYMPTOM

5p5p Mental retardation, cardiac dev. problems

1818pp Physical and mental retardation

XXqq (Fragil(Fragile e X)X) Deformed face, autism

2222qq Thyroid gland/ parathyroid gland development defect

1313qq Tumors, retinoblastoma

INHERITED TRANSLOCATIONSINHERITED TRANSLOCATIONS

TRANSLOCATIONTRANSLOCATION SYMPTOMSYMPTOM

44--20. chromosome20. chromosome mental retardation, deformed face

X X -- 13. chromosome13. chromosome mental retardation




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ACQUIRED DELETIONS IN TUMORSACQUIRED DELETIONS IN TUMORS

DELETIONDELETION TUMORTUMOR

APCAPC--genegene colorectal

Rb (Retinoblastoma) geneRb (Retinoblastoma) gene any tumor

P53 geneP53 gene any tumor

22. chromosome22. chromosome acute myeloid leukemia

TRANSLOCATIONTRANSLOCATION TUMORTUMOR

99--22 chr. (Philadelphia 22 chr. (Philadelphia chromosome)chromosome)

Chronic myeloid leukemia

88--14 chromosome14 chromosome Burkitt - lymphoma

88--21 chromosome 21 chromosome acute myeloid leukemia

ACQUIRED TRANSLOCATIONS IN TUMORSACQUIRED TRANSLOCATIONS IN TUMORS




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most frequent inherited most frequent inherited mental retardationmental retardation

symptoms:symptoms:-- big head, elongated big head, elongated face, big earsface, big ears-- mild mild –– severe mental severe mental retardationretardation

Xq27.3 Xq27.3 ––increased fragilityincreased fragility

X fra(X) fra(X) YX fra(X) fra(X) Y

FRAGILE X SYNDROME

Xq27.3Xq27.3




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A mutation in the FMRA mutation in the FMR--11 gene gene (Xq28) Xq28)

CGG trinucleotidCGG trinucleotid--repeatrepeat expansionexpansion

5’ UTR affected5’ UTR affected

repeatrepeat expansion is followed by methylation and inhibited expressionexpansion is followed by methylation and inhibited expression

FMRFMR--1 protein is a RNA1 protein is a RNA--binding proteinbinding protein

For the mental retardation the For the mental retardation the mGluR5 mGluR5 ((metabotrmetabotropic opic glutamate receptorglutamate receptor) might be) might beresponsible

FRAGILE X SYNDROME




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CGGCGG

CGGCGG

55--50 repeat50 repeat

CGGCGG

5050--200 repeat200 repeat

200 200 -- repeatrepeat

healthyhealthy

‘Pre‘Pre--mutation’mutation’

‘full mutation’‘full mutation’

FMRFMR--1 gene1 geneFRAGILE X SYNDROME




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FISH: FLUORESCENT IN SITU HIBRIDISATION




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FISHFISH




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Direct and indirect methodDirect and indirect method

Excitation lightExcitation light

FluorescenceFluorescence

Fluorescencently Fluorescencently labeled antibodylabeled antibody

NonNon--fluorescently (DIG) fluorescently (DIG) labeled probelabeled probe

Fluorescently labeled Fluorescently labeled probeprobe

Detectable denaturated DNADetectable denaturated DNA




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FISHFISH




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Types of FISH probesTypes of FISH probeschromosome specific probes: for detection of chromosome number abnormalities

X (red) and Y (green) chromosome specific probes in healthy male and female cells




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whole chromosomes are highlighted with the painting probespainting probes – good for detecting translocations

1. chromosome (left), 22. chromosome (middle), 3. chromosome (right) shown with painting probes

Types of FISH probesTypes of FISH probes




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sex chromosome identification with painting probes. two X chromosome (left) in a female cell, a Y chromosome (right) in a male cell.





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the locus/gene specific probes are used to detect any gene of interest, frequently absence or amplification of tumor suppressors of oncogenes

RB1 gene on the 13. chromosome





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Locus/gene specific FISH probesLocus/gene specific FISH probes•• PraderPrader--Willi syndrome 15q11Willi syndrome 15q11--1313•• Angelman syndrome Angelman syndrome 15q1115q11--1313•• DiDi--George syndrome /VCFS 22q11.2George syndrome /VCFS 22q11.2•• Williams syndromeWilliams syndrome 7q11.237q11.23•• WolfWolf--Hirschhorn syndromeHirschhorn syndrome 4p16.34p16.3•• Cri du Chat syndrome Cri du Chat syndrome Xp22.3Xp22.3•• SRY gene Yp11.3 SRY gene Yp11.3 •• XX--linked ichthyosislinked ichthyosis Xp22.3Xp22.3•• Retinoblastoma (RbRetinoblastoma (Rb--gene) 13q14gene) 13q14•• SmithSmith--Magenis syndromeMagenis syndrome 17p11.217p11.2•• MillerMiller--Dieker syndromeDieker syndrome 17p13.317p13.3




4821 trisomy: Down-syndrome

Medical application of FISHMedical application of FISH




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gene specific FISH probes proved amplification of oncogenes (erb-B2, EGFR, myc) and deletion of tumor supressor genes (p53, Rb) in a vast range of tumors

The p53 tumorsuppressor gene shown by gene specific FISH probe in leukaemia cells.





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The Philadelphia chromosome is frequently occuring in chronic myeloid leukaemia (CML). Diagnosis is based on gene specific FISH probes.

Philadelphia chromosome(Ph1)

abl (Abelson cluster region) gene–encodes a tirosin kinase

bcr (breakpoint cluster region)





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A BCR gene is on the 22.,the ABL gene is on the 9. chromosome. Translocation results in fusion of the two genes.





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Telomer specific FISH




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X-CHROMOSOME INACTIVATION

Barr Body rule: #BB = #X-1DOSAGE COMPENSATION

XXX:Two Barr Bodies

Heterochromatinization:

Xist RNA

DNA methylation

Histone methylationCreate PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)




RANDOMDURING EARLY EMBRYOGENESISIRREVERSIBLE





THERE ARE GENES EXPRESSED ON THE INACTIVE X CHROMOSOME!

XX XO

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THE ROLE OF PSEUDOAUTOSOMAL REGIONS (PRESENT ON BOTH X & Y):CHIASMATA FORMATION, TO ENSURE PROPER SEGREGATION OF X & Y CHROMOSOMES




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XX--CHROMOSOME INACTIVATIONCHROMOSOME INACTIVATION--MOMOSSAIAICISMCISM

Anhidrotic Ectodermal DysplasiaCreate PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)



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identify the following genotypesidentify the following genotypes




59Turner syndrome (XO) Turner syndrome (XO)




60Down syndrome (21 trisomy) Down syndrome (21 trisomy)




61Klinefelter syndrome (XXY) Klinefelter syndrome (XXY)




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Normal woman (XX) Normal woman (XX)




63Normal man (XY) Normal man (XY)




cytogenetics eng final [kompatibilitási mód] · the goal of cytogenetics: 1. diagnosis of...

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