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

Karyotyping in PracticeSafrina D. Ratnaningrum

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.

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)

Structure of human chromosomes

▫ Chromosome can be seen clearly during metaphase, maximally condensed

I’m chromosome

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

• Submetacentric

• Metacentricp/shortarm

q/longarm

Chromatid; double/sister chromatid

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

Chromosomal testing

Specimen

• Peripheral blood (anticoagulan agent: HEPARIN)

• Bone marrow aspirates

• Amniotic fluid

• Solid tissue (i.e. POC)

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

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.

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

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

Idiogram: diagrammatic

representation of a karyotype

Metaphase spread Idiogram

Karyotype designation

General principles:

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

Thus,

46, XX Normal female

46, XY Normal male

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)

Numerical and structure abnormality, carcinoma

cells of the lung.

47,XY,+21 (Down syndrome)

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

45,X (Turner syndrome)

47,XXY (Klinefelter syndrome)

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!

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

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)

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

Thank You

Cytovision Genetic ver. 4.5.1