Cytogenetic

Parvaneh AfsharianDepartment of Genetics,

Royan Institute

سيتوژنتيك اصول با آشنایی آموزشي كارگاه برنامهباليني

او�ل روزعنوان زمان

مفاهیم پایه و پیش زمینه

10:45-9

استراحت 11- 10:45

اصول کشت و هاروست

11:15-11

نمونه گیری و کشت(آزمایشگاه)

12:30-11:15

ناهار و نماز 13-12:30

شروع هاروست (آزمایشگاه)

13:30 -13

آماده سازی اسالیدها 13:45-13:30

انجام مراحل فیکس 15-13:45

استراحت 15:20-15

Agingتهیه اسالید و 16:30-15:20

دو�م روز و و اصول مشاوره ژنتیکچک کاریوتایپ ها

10:30-9

استراحت 10:45-3045

اصول روشهای بندینگ 11- 10:45

banding و رنگ آمیزی11-12 (آزمایشگاه)

ناهار ونماز 13-12

سیتوژنتیک بالینی 15-13

استراحت و چک کاریوتایپ 15:30-15

آنالیز کروموزومی 16:30-15:30

DNA Chromosomes

DNA Chromosomes

DNA Chromosomes

Clinical Cytogenetic

History

• Human Chromosomes; 1882 in Tumor cells by Walther Flemming (mitosis discoverer)

Emery & Rimon’s principles and practice of Medical genetics (2007), Elsevier

History

• Chromosome: introduced by von Waldeyer (1888)(colored bodies)

• ≥ 50 years later , 2n= 46

Emery & Rimon’s principles and practice of Medical genetics (2007), Elsevier

History

• 1912: Hans von Winiwater; Spermatogonia (47) & Oogonia (48) (XX/XO system)

• 1922: Painter: 2n= 48 or 46 (XX/XY system)

• 1956: Tjio & Levan (Hypotonic Sol) 2n= 46 in human embryonic cells

Clinical Cytogenetic was bornVon Winiwarter H 1912, Arch BiologiePainter TS 1922 Anat ResPainter TS 1923 J Exp ZoologyTjio JH & Levan A 1956 Hereditas

History

• 1956: Clinical cytogenetics• 1959: +21 (France), 45,XO (UK)• 1960: Ph (t(9;22)) in CML• 1960s end: Banding techniques Chr. Identification• 1977: ISCN

Gilgenkrantz S et al 2003 The history of Cytogenetics. Annales de GenetiqueGarcia-Sagredo JM 2008 Biochim Biophys Acta

نامگذاری المللی بین سیستمانسانی سیتوژنتیک

ISCN : An International System for Human Cytogenetic Nomenclature

المللی بین سیستمنامگذاری

انسانی سیتوژنتیک

Human Chromosomes: Nomenclature & Classification

• Until 1970s: by size & centromer position (Group Analysis, A,B,C,D,E,F,G, Sex chromosomes)

Group Analysis

Sex chromosomes

A

G F

E D

C

B

Definitions

• Cytogenetics– Visual study of chromosomes at microscopic level

• Karyotype– Chromosome complement – also applied to picture of chromosomes

• Idiogram– Stylised form of karyotype

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 on short arm

• Intermediate centromere - submetacentric

chromosome identification

bands numbered from 1, starting near the centromereshort arm on the top, long arm on the bottom

centromere location key in identificationmetacentric – in center; arms about equal in lengthsubmetacentric – arms unequalacrocentric – centromere near one endtelocentric – centromere ‘at one end’

acrocentric – satellites on short arm

Banding techniques

Region Band Sub-Band

2. Sub metacentric

Sub-band

1. Metacentric 3. Acrocentric

1p36.1

GTG banded human chromosomes with banded cartoon along side

Human maleG-banda

• Sampling (Blood: Leukocytes)• Culturing (mitogen; medium; P/S)• Harvesting [colcemid; hypotonic sol (KCl) &

fixative (MeOH:CH3COOH)]• Slid spreading• Staining (aging; banding techniques)

laboratory

Day II

Genetic Counseling

• Medical base• Medical genetic knowledge• Communication process• Deals with the human problems associated with: - occurrence - risk of occurrence - risk of recurrence

of a genetic disorder in a family

• pedigree Medical Genetics for the Modern Clinician, 2006, Lippincott Williams & Wilkins

Pedigree

Inheritance patterns

• Autosomal Dominant Inheritance

• Autosomal Recessive Inheritance

• X-Linked Dominant Inheritance

• X-Linked Recessive Inheritance

Medical Genetics for the Modern Clinician, 2006, Lippincott Williams & Wilkins

Check of your practice

Banding techniques

Chromosome staining

• Q-banding – Quinacrine stain• G-banding – Giemsa stain• C-banding – heterochromatin regions which remain

condensed (regions near centromere are heterochromatin)

• R-banding – reverse banding• FISH – fluorescence in situ hybridization probes for specific genes or locations

probes tagged with fluorescent molecules• Spectral karyotyping

probes specific for each chromosome, different colors

special procedures

C banding – staining of heterochromatin (condensed DNA)region near centromere

High-resolution banding – staining of less condensed chromosome regions non-staining regions on several chromosomes – fragile sites (fragile X – mental retardation)

FISH

Spectral karyotyping

Spectral karyotyping

Array CGH

• Comparative genomic hybridization (numerical Abnormalities)

Clinical

Cytogenetics

Chromosomal

Abnormalities

Chromosomal abnormalities

QNumericalPolyploidy (triploidy, tetraploidy)Aneuploidy (monosomy, trisomy, tetrasomy)

QStructualTranslocationsInversionsInsertionsDeletionsRingsIsochromosomesESAC (Extra Structurally Abnormal Chromosome)

Numerical Chromosomal

Abnormalities

Numerical Chromosomal Abnormalities-I

•Euploidy or polyploidy: multiple N

haploid – 1N or 23 chromosomes diploid – 2N or 46 chromosomes triploid – 3N or 69 chromosomes tetraploid – 4N or 92 chromosomes (~5% spontaneous abortions)

enlarged headfusion of fingers & toesmalformations of mouth,eyes &genitals

Chromosome abnormalities

triploid – 3Ndue to ‘dispermy’ found in 15-18% of spontaneous abortions

PartialHydatidiform mole

Numerical Chromosomal Abnormalities-II

Add/Del. Aneuploidy: 45 or 47 chromosomes

Monosomy: one of a pair missing (usually lethal)

45, X

Trisomy : caused by non-disjunction

XXY (47, XXY); + 21; +18

Aneuploidy of autosomes• Trisomy 21 – Down syndrome (47, XY, +21)

1 in 900 live births leading cause of mental retardation and heart defects

phenotype distinctive skin fold near eye – epicanthic fold spots in iris – Brushfield spots wide skull, flatter than normal at the back tongue often furrowed and protruding congenital heart defects in ~40% of cases physical growth, behavior & mental development prone to respiratory infections leukemia (higher rate than normal)

Maternal age is a factor Medical Genetics for the Modern Clinician, 2006, Lippincott Williams & Wilkins

Down syndrome, trisomy 21

47,XX,+21 or 47,XY,+21

Aneuploidy of autosomes• Trisomy 13 – Patau syndrome (47, XX, +13)

1 in 5,000 live birthscondition lethal

phenotypefacial malformationseye defectsextra fingers or toesmalformations of brain & nervous systemcongenital heart defects

parental age is a factor

Medical Genetics for the Modern Clinician, 2006, Lippincott Williams & Wilkins

Patau syndrome, trisomy 13

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

Incidence at birth 1/5,000

Patau Syndrome

Aneuploidy of autosomes• Trisomy 18 – Edward syndrome (47, XX, +18)

1 in 3,000 live births 80% of live births are female

phenotype small at birth, grow slowlymentally retardedclenched fists; 2nd & 5th fingers overlap 3rd & 4thmalformed feet; heart malformations common

parental age is a factor

Medical Genetics for the Modern Clinician, 2006, Lippincott Williams & Wilkins

Edwards syndrome, trisomy 18

47,XX,+18 or 47,XY,+18

Incidence at birth 1/3,000

Edward Syndrome

Aneuploidy of Sex chromosomes

Abnormalities more tolerated

• Extra X/Y Y: few genes, mostly sex determination X: excess X is inactivated

• Monosomy X, Turners– Majority die during development– Only small proportion survive at birth– Short and infertile– 1 in 2500 live birth

Sex chromosome abnormalities

• Turner Syndrome 45,XO (female)1/2500

• Trisomy X          47, XXX (female)1/1000

• Klinefelter Syndrome 47,XXY (male)1/500

• Extra “Y” chromosome 47,XYY (male)1/1000

Medical Genetics for the Modern Clinician, 2006, Lippincott Williams & Wilkins

Structural Chromosomal

Abnormalities

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

Structural Chromosomal Abnormalities

Dfinitions

• Rearrangements:– Deletion: a segment is lost– Duplication: a segment is doubled– Inversion: a segment within the chromosome is

reversed– Translocation: a segment is moved to a different

chromosome• The origin of these rearrangements can be:

– Breakage and rejoining– Crossing-over between repetitive DNA

Crossing-over between Repetitive DNA

Balanced Rearrangements

Change the chromosomal gene order but do not remove or duplicate any of the DNA of the chromosomes• There are two classes of balanced rearrangements:

1. Inversion: is a rearrangement in which an internal segment of a chromosome has been broken twice, flipped 180 degrees, and rejoined

2. Reciprocal translocation: is a rearrangement in which two chromosomes are each broken once, creating acentric fragments, which then trade places

INVERSIONS

1. Paracentric: if the centromere is outside the inverted segment

2. Pericentric: if the centromere is within the inverted segment

Ordinarily, no genetic material is gained or lost in an inversionThus an individual, whether homozygous or heterozygous for the inversion, generally shows no phenotypic effect.While no genetic material is lost, if breakpoints occur within genes, can cause mutations.

Effects of inversions at the DNA level

However, there are reproductive consequences for the heterozygote.

This is due to problems with pairing of homologues during meiosis.

The homologous chromosomes attempt to align similar regions next to each other as

well as they can.

The chromosomes assume this characteristic loop configuration

This causes no problem, unless crossing over occurs within the inverted region

Crossing-over in paracentric inversion:(inversion does not include the centromere)

Results:

1 normal chromosome

2 deletion chromosomes(inviable)

1 inversion chromosome(all genes present; viable)

Crossing-over in pericentric inversion:(inversion includes the centromere)

Results:

1 normal chromosome

2 deletion/duplication chromosomes(inviable)

1 inversion chromosome(all genes present; viable)

Since the only viable offspring are those that result from gametes which did not

have crossovers within the inverted region, it appears that crossing over in the

inversion has been suppressed

So this is referred to as crossover suppression

Keep in mind that crossing over actually does occur in this region

We just can’t observe the result in the progeny

The genetic result is very tight linkage of genes in an inverted segment

This may be important in the evolution of some, or many, organisms

Why?

A species may evolve a particular set of alleles at several genes on one

chromosome which make individuals possessing them very well adapted.

We call this set a coadapted gene complex

Problem is, every generation meiosis and crossing over threaten to “break up” these

coadapted gene complexes

However, if the coadapted gene complex is within an inversion, no recombination will

occur and the alleles will travel along together generation after generation

TRANSLOCATIONS- Reciprocal- Robertsonian

Usually, the term is used for exchanges of segments between nonhomologous

chromosomes

These are interchromosomaltranslocations

It is also possible to have intrachromosomal translocations,

in which the segment stays within the same chromosome

We will limit our attention to interchromosomal translocations

Within these, there are 2 types: reciprocal (or balanced) and nonreciprocal

As with inversions, translocations usually involve no net gain or loss of genetic material

Because of this, there are usually no phenotypic consequences for being

heterozygous.

Like other chromosomal rearrangements, if breakpoints occur within genes, can

result in mutation of that gene.

The most frequent and important type of translocation is the reciprocal

translocation.

Homozygotes have normal meioses.

Homologues can pair properly, and crossing over poses no problems.

But meiosis is a problem in heterozygotes.

Homologues assume a characteristic cross-shape (cruciform) arrangement at

metaphase

Disjunction can occur in 3 ways, 2 of which produce abnormal gametes

Down Syndrome can arise from a Robertsonian fusion between chromosome 14 and 21.

Most of chromosome 21 is translocated to chromosome 14 - can result in Familial Down syndrome.

Segregation of a Robertsonian Translocation

Gamtes always get either A or B;50% get C. A

BC

Translocations can sometimes be harmful.

Even though there is no gain or loss of genetic material, the change in location of

a segment may alter the regulation of a gene in the segment.

This is especially apparent if the gene is involved in the regulation of cell division.

Lack of proper regulation of such a gene can result in cancer.

In which case, the gene becomes known as an oncogene

A good example is the translocation between chromosomes 9 and 22, creating

the “Philadelphia chromosome”

This causes about 90% of the cases of chronic myelogenous leukemia

Origin of the Philadelphia chromosome in chronic myelogenous leukemia (CML) by a reciprocal translocation involving chromosomes 9 and 22

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Burkitt’s lymphoma is another example of a cancer which is usually (90%) caused by a

translocation (8 and 14)

These are examples of the phenomenon called position effect

The phenotype seen depends not just on the allele of a particular gene, but also the

position of the gene in a particular chromosome

Unbalanced rearrangements

They change the gene dosage of a part of the affected chromosome

• Classes of unbalanced rearrangements:1. All whole chromosome aneuploidies2. Deletions3. Duplications

• The duplicated segment can end up at a different position on the same chromosome, or even on a different chromosome

4. Amplifications

DELETIONS

Deletions involve the loss of a chromosome segment

Because these mutations are due to the loss of genetic material, they cannot revert

to wild type

The effects of the deletion depend on which genes are deleted

And on what alleles of these genes reside on the homologous chromosome

Any genes in the deleted region are now present in a hemizygous condition on the

homologue

If these alleles are recessive, their phenotypes will now be expressed

This phenomenon is calledpseudodominance

A B C D E F

A B E F

WT

deletion

X

a b c d e f

a b c d e f

F1:

A B C D E F All WT

a b c d e f

A B E F

a b c d e f

Mutant phenotype for c and d(c & d phenotype “uncovered by deletion)

&

50%

50%

Deletion Mapping

Several human disorders are due to deletions.

All of these are small deletions - large deletions apparently cannot be tolerated

Also, the deletions have their effects in heterozygotes; homozygotes are probably

lethal

Cri du chat syndrome

Due to a deletion of part of the short arm of chromosome 5

1/50,000 births

Crying babies sound likecats; mental disabilityDeath by about 4 years

DUPLICATIONS

A segment of chromosome is doubled

A good example of duplication is seen in the Bar mutants of Drosophila

Different numbers of copies of the 16A region of the X chromosome

These duplications probably arise by the process of unequal crossing over

Unequal crossing-over produces Bar mutants in Drosophila

As the number of duplicate copies of a segment increases, the likelihood of unequal

crossing over also increases

Thus, once the process has started there is a tendency over evolutionary time for the

number of copies to increase

Duplication in this way by unequal crossing over is thought to be an important process

in the evolution of genes

If a gene is crucial to the organism, it is not free to change much

It certainly cannot take on a new function, since its original one is still needed

But, if a new copy of the gene is produced by unequal crossing over, the extra copy

can evolve over time

Eventually perhaps producing a protein with very different functions

This kind of process can result in “families” of related genes, making similar proteins

Good examples of this are the globin genes, which produce the alpha and beta globin chains which comprise hemoglobin

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

Check of your practice

Chromosome Study(Analysis)

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)

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 5-10 megabases– High resolution (800 bands ; prometaphase chromosome)

– use methotrexate and colchicine

• Dark bands are gene poor

G banding

• Metaphase spreads• Count chromosomes in 10-15 metaphases• If mosaicism suspected, count 30• Detailed analysis of 3-5 metaphases• Used to photograph and cut out• Now computer programmes

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 the 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. Alkali3. Then G band

Chromosome Banding resolutionsTotal bands:

10+X+18q+11pBanding

Resolution

15 35016 37517 40018 41019 42020 43021 44022 450

32-33 55036 610

41 700

CytovisionApplied imaging/Leica