Cytogenetics

Medical Genetic Class, MUMSMohammad R. Abbaszadegan, MT(ASCP), Ph.D., ABMGProfessor of Medical GeneticsHead, Medical Genetics Research Centerabbaszadeganmr@mums.ac.ir8002415

CytogeneticCytogenetics is the study of chromosomes.

The birth of human cytogenetics

• 1956: Tjio and Levan count the full complement of 46 human chromosomes

Definitions

• Cytogenetics– Visual study of chromosomes at

microscopic level• Karyotype

– Chromosome complement – also applied to picture of chromosomes

• Idiogram– Stylised form of karyotype

Clinical indications for chromosomal

analysis• Problems of early growth & development

Include dysmorphic facies, multiple malformation, short stature, ambiguous genitalia, & mental retardation.

• Stillbirth & Neonatal deathIncidence – 10%, live births – about 0.7%

• Fertility problemsi.e. Amenorrhea, couples with history of infertility or habitual abortion,

Incidence of chromosomal abnormalities=3-4%

Clinical indications (cont’)

• Family historyA known or suspected chromosome abnormality in a first degree relative.

• NeoplasiaAll cancers are associated with one or more chromosomal abnormalities.

• Pregnancy in a woman of advanced ageWomen older than 30-35 years

Chromosomes

• Centromere - movement during cell division– divides the chromosomes into short (p) and long

(q) arms

• Telomere - tip of each chromosome– seal chromosomes and retain chromosome

integrity– telomere consists of tandem repeats TTAAGGG– maintained by enzyme - telomerase– reduction in telomerase and decrease in number

repeats important in ageing and cell death

Chromosomes

• Classified according to position of centromere

• Central centromere - metacentric

• Sub-terminal centromere - acrocentric– have satellites which contain multiple copies of

genes for ribosomal RNA

• Intermediate centromere - submetacentric

Chromosomes

Chromosomes

• 22 autosomes and sex chromosomes in pairs• Classified according to:

– Length– position of centromere– presence or absence of satellites

• Chromosomes divided into groups labelled A-G

–A 1-3–B 4-5–C 6-12 + X–D 13-15

–E 16-18–F 19-20–G 21-22 +Y

Karyotyping

• Staining methods to identify chromosomes

• G banding - Giemsa • Q banding - Quinacrine• R banding - Reverse • C banding - Centromeric (heterochromatin)

• Ag-NOR stain - Nucleolar Organizing Regions (active)

Karyotyping – cell preparation

• Need metaphases• Culture cells until sufficient mitotic activity• Add colchicine (or colcemid) to arrest in metaphase

– prevents mitotic spindle fibres forming

• Add hypotonic salt solution to swell cells• Fix with mix of methanol;acetic acid• Want long chromosomes with none overlapping

RPMI 1640FBS (15%)Pen-StrepL-GlutaminePHAPB/BM cells

MTX(10-7M)

Thymidine (10-5M)

PB/BM Culture

3-5 hrs 17 hrs

Harvest5 hrs1) Colcemid (50 g/ml) treatment

2) Hypotonic Solution (KCl;0.075M) treatment3) Fixation (methanol:Acetic acid=3:1)

Slide preparation & Staining (Giemsa-Trypsin)Microscopy and Karyotyping Printing (Photography) and Reporting

Giemsa Banding (G-Banding)

72 hrs

High-resolution Banding TechniqueUsing Methotrexate Cell Synchronization

G banding

• Most common method used• Chromosomes treated with trypsin

– denatures protein• Giemsa stain

– each chromosome characteristic light and dark bands

– 400 bands per haploid genome– Each band corresponds to 6-8 megabases– High resolution (800 bands ; prometaphase

chromosome) – use methotrexate and colchicine

• Dark bands are gene poor

Mazen Zaharna Molecular Biology 1/2009

Chromosome GroupsGroup Chromosomes Description

A 1–3 Largest; 1 and 3 are metacentric but 2 is submetacentric

B 4,5 Large; submetacentric with two arms very different in size

C 6–12,X Medium size; submetacentric

D 13–15 Medium size; acrocentric with satellites

E 16–18 Small; 16 is metacentric but 17 and 18 are submetacentric

F 19,20 Small; metacentric

G 21,22,Y Small; acrocentric, with satellites on 21 and 22 but not on the Y

Q banding

• Used especially for Y chromosome abnormalities or mosaicism

• Similar pattern to G banding – But can detect polymorphisms

• Needs fluorescent microscope

R banding

• Used to identify X chromosome abnormalities

• Heat chromosomes before staining with Giemsa

• Light and dark bands

are reversed

C banding

• Used to identify centromeres / heterochromatin

• Heterochromatic regions – contain repetitive sequences– highly condensed chromatin fibres

• Treat with chromosomes with 1. Acid

2. Alkali

3. Then G band

G-Bands R-Bands C-Bands

Ideogram

Flow karyotyping

Use of flow cytometry to analyze and/or separate chromosomes on the basis of their DNA content.

Flow karyotyping• Hoechst 33258 • Chromomyein A3

ISCN• International System for

Human Cytogenetic Nomenclature

• Each area of chromosome given number

• Lowest number closest (proximal) to centromere

• Highest number at tips (distal) to centromere

Telomeres: 6 bp sequence (ATTGGG) repeated 1000sof times

Centromere: highlyrepetitive satelliteDNA

Short arm: “p” (petit)

Long arm: “q”

p

q

Each arm consists of 2 sister chromatids, each of which represents one molecule of DNA

ISCN

• Normal male– 46,XY

• Normal female– 46,XX

Types of chromosome abnormalities

• Numerical– Aneuploidy (monosomy, trisomy, tetrasomy)– Polyploidy (triploidy, tetraploidy)

• Structural– Translocations– Inversions– Insertions– Deletions– Rings– Isochromosomes– ESAC

Numerical

• Aneuploidy– Autosomal trisomy aneuploidy– Sex chromosomes aneuploidy

• Polyploidy– Whole chromosome set– Triploidy, 69– Tetraploidy, 92

Numerical Chromosome Abnormalities

• Polyploidy - having extra full sets of chromosomes * triploid, tetraploid

• Aneuploidy - An abnormal number of chromosomes

Aneuploidy occurs during cell division when the chromosomes do not separate properly between the two cell

Aneuploidy caused by

• Non-disjunction– failure of homologous chromosomes to separate in

anaphase I – failure of sister chromatids to separate at meiosis II

• Anaphase lag– Chromosomal loss via micronucleus formation

caused by delayed movement of chromosome/chromatid during anaphase• results in daughter cell deficient of that chromosome or

chromatid

NUMERICAL ABNORMALITIES

2n-1 2n 2n+1 2n+2

Polyploidy

Chromosome Nondisjunctions Lead To Monosomies And Trisomies

Chromosome Nondisjunctions Lead To Monosomies And Trisomies

Chromosome Nondisjunctions

Lead To Monosomies And

Trisomies

Nondisjunction can occur during mitosis and be a somatic mutation

Structural

• Breakage in at least 1 chromosome• Translocations

– 2 different chromosomes break and rejoin incorrectly

• Inversions– 2 breaks in same chromosome

• Insertions– Piece of chromosome inserted

• Deletions– Piece of chromosome missing

Structural abnormalities

Duplications And Deletions Can Arise From Unequal Crossovers

Translocations

• Robertsonian – Acrocentric

chromosomes– D and G groups

(13, 14, 15, 21, 22)

• Reciprocal– Any chromosome

Robertsonian

Reciprocal

Reciprocal translocation

• 2:2 segregation– Two chromosomes per gamete– Could produce normal, balanced or unbalanced

gametes

• 3:1 segregation– Three chromosomes to 1 gamete– One chromosome to other gamete– All will be unbalanced

Reciprocal translocation

2:2 segregation• Pachytene

quadrivalent

• Alternate

gives normal or balanced gametes

Reciprocal translocation

2:2 segregation

• Adjacent 1 gives unbalanced

• Adjacent 2 gives unbalanced

Reciprocal translocation

3:1 segregation• Pachytene quadrivalent

• A, C, D together – trisomy for material on C• B alone – monsomy for

material on B

Summary

• 2:2– Alternate A+D or B+C normal or

balanced– Adjacent 1 A+C or B+D

unbalanced– Adjacent 2 A+B or C+D

unbalanced

• 3:1– Three A+B+C or A+B+D

trisomyA+C+D or B+C+D

– One A or B or C or Dmonosomy

Robertsonian Translocations

The p arm of each acrocentric chromosome contains hundreds of copies of the rRNA gene, so losing two p arms does not cause an abnormal phenotype

A Robertsonian translocation is considered a single chromosome, so the karyotype of a male with a Robertsonian translocation involving chromosomes 13 and 14 is written as:

45,XY,rob(13q14q)

The acrocentric chromosomes 13, 14, 15, 21 and 22 form Robertsonian translocations

Structural Chromosome AbnormalitiesRobertsonian Translocations

ISCN 1995International System for Human Cytogenetic Nomenclature

Reciprocal translocation

45,XX,der(13;14)(q10;q10)

Robertsonian translocation

46,XY,t(6;9)(q24;p23)

Rob(14q;21q) Is One Of The More Common Causes Of Down Syndrome

Balanced robertsonian translocation of two 21 chromosomes

der(21;21)

Risk for further offspring

+21fertilization

der(21;21) nulisomic gamete

der(21;21)

m. Downmonosomy 21 – lethal during early prenatal development

Risk: 100%

Reciprocal translocations

• More common than Robertsonian• Break in any chromosome at any point• Phenotypically normal – problems at

meiosis

Structural Chromosome Abnormalities—Reciprocal Translocations

Inversions• Reversal of segment of chromosome

– If too small cannot detect by karyotype – Selected against as would get reduced fertility

• Pericentric– reversed segment includes centromere

• Paracentric– within one chromosome arm

Inversions

Pericentric Paracentric

Inversions outcome after gametogenesis

Insertions

• Segment of 1 chromosome inserted into another

A derA der B

Deletions

• Terminal• Cri du chat, 5p15• Wolf-Hirschhorn, 4p36

• Interstitial• Williams, 7q11.2,• Retinoblastoma, 13q14• DiGeorge, 22q11.2

Cri du Chat (5p-)

• Terminal deletion – 5p15

• Cries like cat

• Mental retardation

DiGeorge syndrome

22q11.2 deletion syndromevelo-cardio-facial syndrome (VCFS)

CATCH-22

Cardiac Abnormality Abnormal faciesThymic aplasiaCleft palateHypocalcemia.

Detection by: fluorescence in situ hybridization (FISH)

Ring chromosome

A derA

Isochromosome

• Two copies of the same arm

• Mirror image around centromere

– Monosomy for 1 chromosome arm – Trisomy for the other arm

Origin of isochromosomesi(Xp)

i(Xq)

Normal separation in anaphase Abnormal division – origin of

isochromosomes Xp and Xq

ESAC

• Extra Structurally Abnormal Chromosome• Abnormal chromosome in addition to 46• Small and difficult to identify• Sometimes called marker chromosomes• Difficult to work out effect on person• May be benign or cause serious mental

handicap

Autosomal & Sex chromosomes numerical abnormality

Down syndrome: history

• First described in the medical literature by Dr. John Langdon Down in England in 1866

• In 1958, Lejeune discovered that the cells from an individual with DS had an extra chromosome 21

Trisomy 21Mental retardationSlanted palpebral fissuresEpicanthal foldsSmall, round, flat faceSmall mouth, protruding Tonguecongenital heart problemsSimian creaseshypotonia, lax joints

Etiology of Down Syndrome

• 95% - Free Trisomy 21– Don’t need to check the parents’ karyotypes

• 4% - Unbalanced Translocation (must check parental karyotypes):– 3% de novo– 1% familial

• Most frequently due to rob 14q21• Adjacent segregation

• 1% Mosaicism

Chromosomes of gametes that theoretically can be produced by a carrier of a Robertsonian translocation

Robertsonian translocation 14q21q

45.XX,rob(14;21)

Some genes located on the long arm of chromosome 21

Down's syndrome critical region (DSCR)

NFATc (for 'nuclear factor of activated T cells')

Trisomy 18

• Mental retardation• Growth retardation• Short neck• Cleft lip/palate• Dislocated• Hips/abnormal pelvis• Deformed, low-set ears

• Hypertonia• Congenital heart disease• Horseshoe kidneys• Hydronephrosis• Short sternum• Pyloric stenosis

Edward syndrome, Trisomy 18

Edwards syndrome, trisomy 18

Trisomy 13

• Mental retardation• Growth retardation• Microcephaly • cleft lip/palate• Small jaw (micrognathia)• Low-set ears

• Polydactyly • Clenched over hanged finger• Congenital heart defects• rocker bottom feet• Seizures• Low birth weight

Patau syndrome, trisomy 13

47,XX,+13 or 47,XY,+13

Incidence at birth 1/5,000

Patau syndrome, Trisomy 13

sex chromosome aneuploidies

Because of X inactivation and because of the paucity of genes on the Y chromosome, aneuploidies involving the sex chromosomes are far more common than those involving autosomes.

Nondisjunction of sex chromosomes during spermatogenesis – 1st meiotic division

XY

XY

XY XY

XXY XXY X X+X

nondisjunction

fertilization

Nondisjunction of sex chromosomes during spermatogenesis – 2nd meiotic division

XY

YX

XX Y Y

XXX X XY XY+X

nondisjunction

fertilization

Nondisjunction of sex chromosomes during spermatogenesis – 2nd meiotic division – Y chromosome

XY

YX

X X YY

XX XX XYY X+X

nondisjunction

fertilization

Turner syndrome 45,X

99% of Turner syndrome embryos are spontaneously aborted.

Individuals are very short, they are usually infertile. Characteristic body shape changes include a broad chest with widely spaced nipples and may include a webbed neck.

IQ and lifespan are unaffected.

Testes are small and fail to produce normal levels of testosterone which leads to breast growth (gynaecomastia) in about 40% of cases and to poorly developed secondary sexual characteristics.

There is no spermatogenesis. These males are taller and thinner than average and may have a slight

reduction in IQ. Many Kleinfelter males lead a normal life. Very rarely more extreme forms of Kleinfelter's syndrome occur where

the patient has 48, XXXY or even 49, XXXXY karyotype. These individuals are generally severely retarded.

Kleinfelter’s syndrome

karyotype Barr body47 XXY 2n+1 148,XXXY 2n+2 248,XXYY 2n+2 149,XXXXY 2n+3 350,XXXXXY 2n+4 4

Kleinfelter’s syndrome

sex chromosome aneuploidies47,XYY males

Males are tall but normally proportioned.

XXX females It seems to do little harm, individuals are fertile and do not

transmit the extra chromosome.They do have a reduction in IQ comparable to that of Kleinfelter's

males.

Parental origin aneuploidy gamet

Mosaicism

Definition: Mosaicism describes the occurrence of cells

that differ in their genetic component from other cells of the body.

Presence of two populations of cells with different genotypes in one individual who has developed from a single fertilized egg

Mosaicism Mosaicism

Germline: affecting only egg or sperm cells Somatic: affecting cells other than egg or

sperm cellsUsually caused by a post zygotic mutation

Combination of both.

Germline Mosaicism

A person with a germline mosaicism Will not be affected with the disorder caused by

the mutation because the mutation is not in the other cells of the body.

Therefore they are unaware that they possess this germline mutation

Genetic testing using blood or tissue samples (other than germline tissue) from individuals who only have a germline mutation will be negative for the mutation.

Germline mosaicism can be observed with any inheritance pattern, but it is most commonly seen with autosomal dominant and X-linked disorders.

Usually, when unaffected parents have a child with an autosomal dominant mutation—de novo mutation/sporadic mutation

In rare situations, unaffected parents can have more than one child with an AD disorder. This can be caused by germline mosaicism.

Germline mosaicism has been observed in a number of conditions, including Achondroplasia Osteogenesis imperfecta Duchenne muscular dystrophy.

Somatic Mosaicism

An individual with a somatic mutation will express the phenotype of that mutation depending on how many and which cells are affected.

Typically, individuals with somatic mosaicism exhibit a milder phenotype since only a proportion of cells contain the

mutation because the mutation is confined to a finite

segment of the body.

Confined placental mosaicism

discrepancy between the chromosomal makeup of the cells in the placenta and the cells in the baby.

Trisomic cells are detected on chorionic villus sampling and only normal cells are found on a subsequent prenatal test, such as amniocentesis or fetal blood sampling

Molecular Cytogenetic

Molecular Cytogenetics Fluorescence In Situ Hybridization

• Metaphase FISH

• Interphase FISH

• Fiber FISH

Spectral Karyotyping

Chromosome sorting and microdissection

Comparative Genomic Hybridization Array CGH

FISH (fluorescence in situ hybridization)

To detect and localize the presence or absence of specific DNA sequences on chromosomes

FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence similarity

Fluorescence microscopy

FISH (fluorescentie in situ hybridisatie)

Type of FISH ProbeCentromere specific Probe highly repetitive alpha satelliteTelomere specific Probe Subtle chromosome abnormality that involve ends of chromosomeLocus-specific Probe Detect structural abnormalityWhole chromosome paint Probe

Application of FISH

Revolutionized cytogenetic analysis:• Submicroscopic chromosome abnormalities

– Microdeletion syndromes– Telomeric rearrangements

• Marker chromosome identification• Interphase analysis

– Rapid prenatal Diagnosis

Interphase FISH Analysis

• Interphase FISH can give information about chromosome number in non-cycling cells

– Uncultured cells– Patients on chemotherapy (after chemotherapy there is no cycling cells)

• Time saving since standard cytogenetics is dependent on obtaining actively dividing cells after culturing

Metaphase- FISH

Interphase-FISH

Prenatal Aneuploid screening by Interphase FISH

• Trisomies for chromosome 13, 18, 21 and sex chromosomal aneuploidy

(quick analysis in two days but not in musaism we need to be wait for CVS culture results for full G-band results

• Probes generally target the centromeres • Takes 2 days for results• Full G-banded karyotype takes 7-14 days

DNA probes for chromosomes 13, 18, 21, X, and Y

Since there are two signals each for chromosomes 13, 18, and 21 and one signal each for the X and Y chromosome this fetus is a normal male with respect to the Aneuploid Screen test.

The nucleus on the left has been hybridized to probes for chromosomes 13 (green), and 21 (red).

The nucleus on the right has been hybridized to probes for chromosomes 18 (aqua), X (green), and Y (red).

Normal Down syndrome

Green: Chromosome 13Red: Chromosome 21

131

chromosome orientation and direction fluorescence in situ hybridization COD-FISH

A new approach for detecting chromosomal inversions. 

single-stranded probes to one, and only one, chromatid of a metaphase chromosome.

An inversion becomes detectable as a "switch" in probe signal from one chromatid to the other

Spectral Karyotype of human chromosomes

CGH (Comparative genomic hybridization) Is a molecular-cytogenetic method for the analysis

of copy number changes (gains/losses) - often in tumor cells

CGH

CGH

advantages

whole genome in 1 experiment

no need to culture tumor cells

sensitive detection of gene amplification

disadvantages

limited resolution (~10 Mb del/dup)

laborious

only gains and losses / no balanced rearrangements

no information on the nature of the aberrations

retrospective analysis

CGH (Comparative genomic hybridization)

CGH will detect only unbalanced chromosomal changes. Structural chromosome aberrations such as - Balanced reciprocal translocations - Inversions cannot be detected, as they do not change the copy number.

Array comparative genomic hybridization

Is a technique to detect genomic copy number variations at a higher resolution level than chromosome-based comparative genomic hybridization (CGH).

Efficiency a-CGHUsing this method, copy number changes at a level of 5-10 kilobasesof DNA sequences can be detected.

Today even high-resolution CGH (HR-CGH) arrays are accurate to detect structural variations at resolution of 200 bp

This method allows one to identify new recurrent chromosome changes such asMicrodeletions Microduplications

Karyotyping (G-banding)

Provides a global view of metaphase chromosomal characteristics (number, type, shape etc)

Each chromosome has a characteristic banding pattern that helps to identify them

Spectral Karyotyping (SKY)

Allows simultaneous visualization all the chromosomal pairs in different colors using chromosome specific probes

More accurate than G-banding

Fluorescent in situ Hybridization (FISH)

More specific and sensitive than karyotyping Uses fluorescent probes to detect and localize the presence or absence of specific DNA sequences on chromosomes Resolution: 5 Mb – Metaphase 2 Mb – Interphase 0.5 Mb – Fibre FISH