karyotyping in practice - safrinadewi.lecture.ub.ac.id · karyotyping •greek karyon = seed,...

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Karyotyping in Practice Safrina D. Ratnaningrum

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Page 1: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:

Karyotyping in PracticeSafrina D. Ratnaningrum

Page 2: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:

Genetic testing

• Chromosomal level▫ Karyotyping, >4 Mbp▫ CGH (comparative genomic hybridization), 3-10

Mbp High resolution CGH Microarray CGH

▫ Multicolor FISH (M-FISH)▫ FISH

• Molecular level (DNA testing) Depend on the purpose/disease will be tested.

Page 3: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:

Karyotyping

• Greek karyon = seed, nucleus

• Karyotype: an organized profile

of a persons chromosomes.

• Karyotyping: a test to examine chromosomes in a sample of cells, which can help identification and detection if any abnormality establish the diagnosis of genetic diseases genetic counseling (i.e. recurrence risk, further management)

Page 4: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:

Structure of human chromosomes

▫ Chromosome can be seen clearly during metaphase, maximally condensed

Page 5: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:

I’m chromosome

Page 6: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:

Type of chromosome• Acrocentric: 13, 14, 15, 21, 22

• Submetacentric

• Metacentricp/shortarm

q/longarm

Chromatid; double/sister chromatid

Page 7: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:

Chromosome number and morphology

Chromosome are ordered of decreasing length (except chr 21 is shorter than 22)

• Chromosome autosomes: 1-22

• Chromosome sex: X and Y

Figure. Karyogram, 46,XY

Page 8: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:

Chromosomal testing

Page 9: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:

Specimen

• Peripheral blood (anticoagulan agent: HEPARIN)

• Bone marrow aspirates

• Amniotic fluid

• Solid tissue (i.e. POC)

Page 10: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:

Stage Process Reagent

Cell culture Cell culture FBS+PHA (mitogen)

Harvesting Mitotic arresting Colchicine/Colcemid

Cells breaking KCl 0,075 M

Droping Chromosome fixation Carnoy’s (acetic acid+methanol, 1:3)

Staining Chromosome staining (Non-banding; Banding)

Karyotiping Manual; Cytovision Software ver.4.5.1

Page 11: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:

Chromosome staining

• Solid (non-banding) staining

• Banding techniques:

▫ G-banding (Giemsa) G-bands

▫ R-banding (reverse bands of G-bands)

▫ C-banding to stain centromer (identify Y chrwithin acrocentric chr; sex determinant)

▫ Silver banding to stain the satellites of acrocentric chr.

Page 12: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:
Page 13: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:

Karyotype

• G-staining G-bands▫ The light bands on

chromosome regions rich in GC and genes.

▫ Dark bands rich in AT and few on genes. Ex: Chromosome 19, dense with genes, has few dark bands.

▫ Region – Bands –Sub bands

Result: 46,XX

Page 14: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:

Fig. Resolution level of banding. From left 300. 400, 550, 700, 850.

Page 15: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:

Idiogram: diagrammatic

representation of a karyotype

Metaphase spread Idiogram

Page 16: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:

Karyotype designation

General principles:

Total number chr, chr sex, abnormality (if any)

Thus,

46, XX Normal female

46, XY Normal male

Page 17: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:

Chromosome level mutationAbnormalities of chromosome:Numerical

Polyploidy: >2n (normal = diploidy/diploid, 2n, 46)i.e. Triploidy (3n); tetraploidy (4n)

Aneuploidy: loss or gain of one or more chromosomes Autosomal chromosome, monosomy (lethal), trisomy (i.e.

trisomy 21, trisomy 13)

Sex chromosome, monosomy (45,X), trisomy (47,XXY)

Structural (i.e. translocation, ring chromosome, deletion, duplication,

inversion, isodisomy)

Page 18: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:

Numerical and structure abnormality, carcinoma

cells of the lung.

Page 19: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:

47,XY,+21 (Down syndrome)

Page 20: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:

Physical featuresMore common physical features are:• Hypotonia• Upslanting palpebral fissures• Small mouth with protruding tongue• Single transverse palmar crease• Short finger and short stature• Short fifth finger with clinodactily• Sandal gap• Flat nasal bridge• DS is the most common genetic cause of

mental retardation in human• Mentally retarded is variable:

▫ Mild (IQ: 50-70) to moderate (IQ: 35-50)▫ Occasionally severe (IQ: 20-35%)

20

Page 21: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:

45,X (Turner syndrome)

Page 22: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:

47,XXY (Klinefelter syndrome)

Page 23: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:

DS caused by translocation

46,XX,der(14;21)(q10;q10)

Both are result of Robertsonian translocation with different consequence of the recurrence risk for next pregnancy!

Page 24: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:

46,XY,t(9;22)(q34;q12) (CML)

Page 25: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:

CML (Chronic Myeloid Leukemia)

• The reciprocal translocation fuses the phl gene a combination between Abl1 gene on chr 9 and BCR gene on chr 22 elongated chr 9 and truncated chr 22 (the Philadelphia chromosome)

Page 26: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:

Mosaicism in karyotyping result

• Mosaicism is individual with >1 cell lines.

• Cell counting will be increased to 50-100 cells clusters, examples:

mos 47,XY,+21/46,XY

mos 45,X/46,XX

Page 27: Karyotyping in Practice - safrinadewi.lecture.ub.ac.id · Karyotyping •Greek karyon = seed, nucleus •Karyotype: an organized profile of a persons chromosomes. •Karyotyping:

Thank You

Cytovision Genetic ver. 4.5.1