lsm4225-1 cytogenetics
DESCRIPTION
LSM4225 Genetic medicine in the post-genomic eraTRANSCRIPT
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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
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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.
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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
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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
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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
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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.
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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.
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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.
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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.
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Karyotype
The chromosomes from a metaphase spread (left) are rearranged to form a pictorial representation called a karyotype (right).
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Classes of Chromosomes (based on centromere position)
metacentric submetacentric
acrocentric
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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)
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G-Banded Metaphase Spread
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Group A: Group B:
Group C:
Group D: Group E:
Group F: Group G:
A Male Karyotype
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Chromosome Idiogram
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Normal and High Resolution (HR) Chromosome Banding
400 bands* 550 bands* 850 bands* (HR)
*numbers are total bands per haploid set
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Chromosomal Abnormalities Can be classified according to type and origin
Types of abnormalities Numerical abnormalities Structural abnormalities
Origin of abnormalities Constitutional Acquired
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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
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Reciprocal Translocation in Chronic Myeloid Leukemia
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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).
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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.
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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.
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Monosomy 7 in Childhood Myelodysplastic Syndrome (MDS)
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Reciprocal (9;11)(p22;q23) Translocation in Acute Myeloid
Leukemia (AML M5)
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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)
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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.
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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
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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
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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.
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Triploidy and Tetraploidy
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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/
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Trisomy 21, Down Syndrome (47,XX,+21)
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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
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Down Syndrome
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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%
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Down Syndrome due to (Robertsonian) Translocation
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Pairing and segregation
Parental Origin of Robertsonian Translocation Down Syndrome
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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.
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Trisomy 13, Patau Syndrome (47,XX,+13)
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Patau Syndrome
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Trisomy 18, Edward Syndrome (47,XX,+18)
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Edward Syndrome
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Monosomy X, Turner Syndrome (45,X)
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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.
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Turner Syndrome
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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.
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Turner Variant due toMosaic 45,X and 46,X del(X)(p11)
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Turner Variant due toMosaic 45,X and 46,X i(X)(q10)
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Turner Variant due toMosaic 45,X and 46,X idic(X)(p11)
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Klinefelter Syndrome (47,XXY)
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Klinefelter Syndrome
Also known as Testicular Dysgenesis
tall, thin habitus
delayed puberty
gynaecoid habitus
hypogonadism
infertility
Usually due to 47,XXY
Sometimes 49, XXXXY
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Patient with tall stature
Jacob Syndrome (47,XYY)
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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
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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.
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Reciprocal Translocation
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Robertsonian Translocation
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Robertsonian Translocation
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Chromosome Deletion
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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
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Outcome of Intrachromosomal Breaks
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Dysmorphic baby with a 46,XY, r(22)(q13p13) Karyotype
Ring Chromosome
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Insertion/Deletion vs. Duplication
Duplication
InsertionDeletion
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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