LSM 4225Applications of Genetics in

Medicine: Cytogenetics - Karyotyping

A/P Samuel S. ChongDepartment of Pediatrics, Yong Loo Lin School of Medicine,

National University of SingaporeDepartment of Laboratory Medicine, National University Hospital

samuel_chong@nuhs.edu.sg

Genetic Testing & Screening

Genetic Testing: analysis of human DNA, RNA, chromosomes, proteins, and certain metabolites in order to detect (heritable) disease related genotypes, mutations, phenotypes or karyotypes for clinical purposes. Includes screening for mutations. Purpose:

Confirmation of clinical diagnosis of a symptomatic individual

Prenatal diagnostic testing

Predictive testing, e.g. Huntington disease

Carrier testing

Newborn testing

Susceptibility testing, e.g. cardiovascular disease

Forensic/identity testing

Genetic Screening: usually a population screen to identify asymptomatic people at an increased risk of particular adverse outcome.

Types of Genetic Testing Biochemical

protein assay

enzyme assay

antigen/antibody immunoassay.

Cytogenetics chromosome analysis (karyotyping).

Molecular Cytogenetics FISH (fluorescence in situ hybridization)

M-FISH (multi-color FISH) or SKY (spectral karyotyping)

M-BAND (multi-color chromosome banding)

CGH (comparative genomic hybridization)

Molecular Diagnostics Southern/dot-blot hybridization

PCR

Cytogenetics and the Human Karyotype

Chromosomal Abnormalities

FISH (fluorescence in situ hybridization)

Whole Chromosome Painting

Molecular Karyotyping (mFISH/SKY and mBAND)

Comparative Genomic Hybridization (CGH)

Array CGH, SNP arrays

Cytogenetics

Cytogenetics, Chromosomes, & DNA

Cytogenetics is the study of chromosomes and their abnormalities.

Chromosomes are a temporary state of the DNA in the nucleus

The DNA is highly condensed (super coiled) in the chromosomes

The double helix is the basic 3D structure of the DNA

The basic building stones of the DNA are the bases

Human ChromosomesThe number of chromosomes in human cells is 46, or 23 pairs

44 autosomes and

2 sex chromosomes

Females have 2 Xchromosomes

Males have an X and aY chromosome

Each chromosome consists of a very long strand of DNA molecule that is packaged with associated proteins.

The Cell Cycle & Metaphase

Interphase:the "holding" stage. 90% of a cell's cellular cycle may be spent in interphase.

Prophase:in the beginning of prophase the condensed, X-shaped chromosomes are visible.

Metaphase:the chromosomes line up in the middle of the cell for being divided equally into the daughter cells.

Anaphase & Telophase:the cell finishes the chromosome separation and the division of the cell.

Karyotyping Karyotyping is the examination of chromosomes to identify

genetic abnormalities, either in chromosome count (numerical aberration) or in chromosome structure (structural aberration).

Chromosomes are usually very extended between cell cycles (chromatin).

To visualize them, we make use of the knowledge that chromosomes are most condensed in the metaphase of mitosis.

A drug (colchicine) is used to disrupt spindle formation. This prevents the mitotic cell from progressing to anaphase, thus arresting them in metaphase.

Steps in Karyotyping

Sample culture: peripheral blood lymphocytes, skin fibroblasts, bone marrow, amniocytes or chorionic villi.

Cell-cycle arrest at metaphase: add colchicine to prevent spindle formation.

Cell swelling and fixing: add hypotonic saline, then methanol:acetic acid mixture.

Chromosome banding: several methods can be used to stain the metaphase chromosomes so that we can identify them by size, centromere position and banding pattern.

Karyotype

The chromosomes from a metaphase spread (left) are rearranged to form a pictorial representation called a karyotype (right).

Classes of Chromosomes (based on centromere position)

metacentric submetacentric

acrocentric

Structure of a Chromosome

Centromere:Contains specific DNA sequencesEssential for segregation during cell division

Telomere:Specific DNA sequences found at the end of the chromosomes Maintains the integrity of chromosomeEnsures complete replication of the ends of the chromosomesHelp establish chromosome pairing

Telomere

p arm(short arm)

q arm(long arm)

Dark bandLight band (gene-rich)

G-Banded Metaphase Spread

Group A: Group B:

Group C:

Group D: Group E:

Group F: Group G:

A Male Karyotype

Chromosome Idiogram

Normal and High Resolution (HR) Chromosome Banding

400 bands* 550 bands* 850 bands* (HR)

*numbers are total bands per haploid set

Chromosomal Abnormalities Can be classified according to type and origin

Types of abnormalities Numerical abnormalities Structural abnormalities

Origin of abnormalities Constitutional Acquired

Acquired Abnormalities

Acquired: i.e. born with normal chromosomes but acquired abnormal chromosome(s) along the way

Etiology: problem/mistake during mitosis

Types of chromosomal abnormalities seen

Numerical

Structural

Clinical Spectrum: Many present as cancers

Leukemia

Solid tumours

Reciprocal Translocation in Chronic Myeloid Leukemia

Chronic Myeloid Leukemia (CML) Characterized by replacement of the bone

marrow with malignant, leukemic cells.

Usually diagnosed by finding a specific structural chromosomal abnormality called the Philadelphia Chromosome (Ph).

Ph is an abnormally short chromosome formed by a translocation between chromosomes 22 and 9.

This was the first consistent chromosome abnormality found in any kind of malignancy.

Etiology of CML:

Mitotic error in a single bone marrow cell.

Gave rise to leukemia through clonal expansion (the production of many cells from a single cell).

Molecular Basis of CML The BCR-ABL Fusion Oncogene

ABL and BCR are normal genes found on chromosomes 9 and 22, respectively.

After translocation, two fusion genes are generated:

BCR-ABL on the Ph chromosome.

ABL-BCR on the derivative chromosome 9.

The BCR-ABL fusion gene produces excessive abnormal tyrosine kinase.

This leads to uncontrolled cell growth, giving rise to cancer.

This is also the pathogenetic basis of some other leukemias.

Designer Drugs Targeting CML Making use of this knowledge, a designer drug Imatinib

mesylate or Gleevec was created.

Gleevac is a tyrosine kinase inhibitor.

It works by binding to the abnormal BCR-ABL protein (which is a receptor) and blocks ATP binding.

Without the energy provided by the ATP molecule, the BCR-ABL protein cannot function.

Gleevec therefore induces apoptosis in cancerous cells and inhibits tumor growth.

However, since the binding is dependent on the specificity of the protein, acquired mutations in the fusion gene that alter the binding of the drug to the protein can give rise to resistance to this drug.

Monosomy 7 in Childhood Myelodysplastic Syndrome (MDS)

Reciprocal (9;11)(p22;q23) Translocation in Acute Myeloid

Leukemia (AML M5)

Constitutional Abnormalities Constitutional: i.e. born with abnormal chromosome(s)

Etiology: problem/mistake during oogenesis or spermatogenesis, abnormal fertilization, or other first mitotic event in the zygote.

Types of chromosomal abnormalities seen

Numerical or structural

Examples

Trisomies 21 (Down syndrome), 13 (Patau syndrome), 18 (Edward syndrome)

Monosomy X (Turner syndrome)

DiGeorge syndrome (microdeletion in chromosome 22q11)

Clinical Spectrum of Constitutional Chromosomal Abnormalities

Individual suffers from infertility but is otherwise healthy

2-4% of infertile couples have a chromosomal abnormality.

Fetal demise (miscarriage) or stillbirth

15% of pregnancies end in miscarriages. Half of these are due to chromosomal abnormalities.

5% of stillborn babies have a chromosomal abnormality.

Abnormal baby at birth

0.7% of newborns have chromosomal abnormalities.

May have features such as malformations, developmental delay, failure to thrive.

Chromosome Defects at BirthNumerical abnormalities:

Trisomy 21 (Down): 1:800 (1:100 at mothers age 40)

Trisomy 18 (Edward) and Trisomy 13 (Patau): 1:4000

Monosomy X (Turner): 1:2500

Jacob Syndrome (XYY): 1:2000

Structural abnormalities:

Cri-du-Chat (deletion of chromosome 5p terminal) 1:50000

Pallister Killian (partial duplication of chromosome 12p)

Angelman (partial deletion of mothers chromosome 15) 1:15000

Prader-Willi (partial deletion of fathers chromosome 15) 1:15000

Numerical Abnormalities

Abnormal number of chromosomes but each chromosome is normal Gain or loss in one or two chromosomes (aneuploidy) Gain of a complete haploid set of chromosomes

(polyploidy) Mixture of two or more different cell lines (mixoploidy)

Etiology: Failure of chromosomes or sister chromatids to

separate correctly (nondisjunction) Can occur during meiosis or mitosis

Consequences of Numerical Abnormalities

Results in loss or gain of genes, thus perturbing the balance in gene expression.

This can lead to cellular dysfunction (e.g. uncontrolled growth, abnormal organs/malformations) or cell death (e.g. miscarriages).

Generally,

Gain is better tolerated than loss.

Abnormalities of autosomes have more serious consequences than similar abnormalities involving the sex chromosomes.

Triploidy and Tetraploidy

Constitutional Numerical Abnormalities

The main factor influencing the risk of constitutional numerical chromosomal abnormalities is maternal age

Evidence for other factors such as environment, genetic susceptibility is not strong

From www.aafp.org/

Trisomy 21, Down Syndrome (47,XX,+21)

Down Syndrome Most common autosomal trisomy

80% of affected conceptions do not survive to term (20% do!)

Overall incidence in liveborn infants is 1:650

Most common genetic cause of mental retardation

Clinical Features

Face: Epicanthal folds, upslanting eyes, flat nasal bridge

Hands: Simian creases, clinodactyly (curved finger)

Feet: Sandal gap toes

CNS: Developmental delay, risk for early onset dementia, partial dislocation of C1/C2 vertebrae

Heart: Congenital heart defects

Abdomen: Duodenal atresia (lack of an opening)

Increased risk of leukemia in young adults

Alzheimers disease in middle age

Down Syndrome

Down Syndrome Origins and Risks

Maternal age at delivery Risk

All ages combined 1:650

20 y 1:1420

30 y 1:1140

35 y 1:360

40 y 1:100

45 y 1:30

Origin: Meiotic Non-dysjunction 92%Translocation 3 4%Mosaic 2 4%

Down Syndrome due to (Robertsonian) Translocation

Pairing and segregation

Parental Origin of Robertsonian Translocation Down Syndrome

Down Syndrome due to Mosaicism

A mosaic DS child has two populations of cells,

the trisomy 21 cells

and a second cell line, usually a normal cell line (likely due to spontaneous loss of one chr. 21.

The physical features may be milder in these individuals, particularly if there is a large proportion of normal cells.

Trisomy 13, Patau Syndrome (47,XX,+13)

Patau Syndrome

Trisomy 18, Edward Syndrome (47,XX,+18)

Edward Syndrome

Monosomy X, Turner Syndrome (45,X)

Turner Syndrome One of the most common causes of fetal hydrops (body

cavities filled with fluid and soft tissue is edematous).

99% of Turner fetuses abort spontaneously; 1% survive.

Incidence: 1 in 2,500 females.

Distinct cystic hygromas (due to failure of lymphatics to form and drain properly) are a common finding in affected fetuses.

Newborn may have lymphedema of hands and feet, coarctation (constriction) of the aorta, neck webbing.

Older children may have short stature, delayed puberty, infertility, neck webbing, cubitus valgus (deviation of extended forearms outwards), and other congenital anomalies (heart, kidney).

Intelligence is normal although some girls have learning disability.

Turner Syndrome

Causes of Turner Syndrome Constitutional Monosomy X (45,X)

Accounts for ~50% of cases Mosaic for Monosomy X

Mixture of 45,X cell line and either 46,XX, 46,XY, or (rarely) 47,XXX cell line

Accounts for ~50% of cases Mosaic for X chromosome abnormalities (rare)

Includes isochromosome X, isodicentric X, and partial deletion of one X chromosome

Important to determine the cause because: The risk of malignant tumor (gonadoblastoma) is higher

in mosaics with a cell line containing a Y chromosome. The clinical features may be milder in the mosaic. Growth hormone is an effective treatment for the short

stature.

Turner Variant due toMosaic 45,X and 46,X del(X)(p11)

Turner Variant due toMosaic 45,X and 46,X i(X)(q10)

Turner Variant due toMosaic 45,X and 46,X idic(X)(p11)

Klinefelter Syndrome (47,XXY)

Klinefelter Syndrome

Also known as Testicular Dysgenesis

tall, thin habitus

delayed puberty

gynaecoid habitus

hypogonadism

infertility

Usually due to 47,XXY

Sometimes 49, XXXXY

Patient with tall stature

Jacob Syndrome (47,XYY)

Structural Abnormalities Common chromosome rearrangements include

Deletion (interstitial or terminal) Inversion (paracentric or pericentric) Duplication Insertion (interstitial or terminal) Translocation Ring Marker

Etiology: Problem during meiosis or mitosis, e.g.

Breaks in chromosomes Unequal exchange during crossovers Failure of centromeres to separate correctly

Consequence of Structural Abnormalities

There may be a loss or gain of genes, thus perturbing the balance in gene expression.

The break in the chromosome may alter the expression or change the product of a gene.

These can lead to cellular dysfunction (e.g. uncontrolled growth, abnormal organs/malformations) or cell death (e.g. miscarriages)

Some structural abnormalities are clinically benign if:

There is no gene disruption

There is no loss or gain of gene copy number

The loss/gain involves multi-copy genes, e.g. rDNA genes.

Reciprocal Translocation

Robertsonian Translocation

Robertsonian Translocation

Chromosome Deletion

Cri-du-Chat Syndrome(5p- Syndrome)

Terminal deletion of the short arm of chromosome 5

Also known as 5p syndrome

Clinical Features

Dysmorphism

Cat-like cry

Mental retardation

Congenital heart abnormalities

Outcome of Intrachromosomal Breaks

Dysmorphic baby with a 46,XY, r(22)(q13p13) Karyotype

Ring Chromosome

Insertion/Deletion vs. Duplication

Duplication

InsertionDeletion

Recurrence Risk of Constitutional Chromosomal Abnormalities

Spontaneous

Low recurrence risk.

Parental numerical abnormality

Up to 50% recurrence risk, although the affected parent may be infertile/subfertile

Parental structural abnormality

Exact risk is dependent on the type of chromosomal abnormality involved.

There is also a risk of recombination of the structural abnormality during meiosis.

1-2% risk in translocations

15-20% risk in pericentric inversions