Download - From chromosome to gene analysis
From chromosome to gene analysis
Part I
A chromosomal mutation vs. a genetic mutation
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Variations in the human genome range from single nucleotide changes to wholechromosomal aneuploidies
Cytogenetics
Chromosomal analysis
Chromosome types
Chromosome types in human:
Metacentric
Submetacentric
Akrocentric
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Human chromosome groups A 1-3 big metacentric chromosomes
B 4-5 big submetacentric chromosomes
C 6-12 and X medium submetacentric chromosomes
D 13-15 big acrocentric chromosomes
E 16-18 small submetacentric chromosomes
F 19-20 small metacentric chromosomes
G 21-22 and Y small acrocentric chromosomes
A B
C
D E
F G
Polyploidy Aneuploidy
Triploidy
3nTetraploidy
4n
Trisomy
2n +1
Monosomy
2n - 1
Numerical abnormalities of
chromosomes
Triploidy69,XXX 69,XXY 69,XYY
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Monosomy45,X
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Trisomy47,XX+13, 47,XX+18, 47,XX+2147,XY+13, 47,XY+18, 47,XY+21
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47,XXY; 47,XXX; 47,XYYI 47,XXY= Klinefelter Syndrome
II 47,XXX = Triple X Syndrome
10III 47,XYY = Jacob Syndrome
Structural aberration
Balanced Unbalanced
Translocation
Inversion
Deletion
Duplication
Ring chromosome
Isochromosome
Marker chromosome
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Genetic testing:
• The analysis of human DNA, RNA, chromosomes, and certain metabolites
• In order to maximize the benefits of genetictesting, it is essential to target the best test to the patient
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• The clinician must use all of the clinical information to create a differential diagnosis
• In can be made by means including physical examination, medical&family history, radiological stydy, utrasound imaging…
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• Initially, genetic testing was primary biochemical
• Since 1959, when J.Lejeune&P.Jacobs discovered the chromosomal cause of Down syndrome, advances of cytogenetics have led to new methods of genetic testing:
banding
and later fluoroscence in situ hibridization
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„Hate the disease, love the patient: That is the practise of
medicine” J. Lejeune
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What test to order?No single technology will find all mutations types (yet…)
• Consider differential diagnosis
• Consider expected mutation types
• Known/suspected clinical diagnosis
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Classification of genetic testing
Diagnostic Predictive
Presymptomatic
Predispositioning
Carrier
Newborn17
Types of genetic testing• Karyotyping&Banding chromosomes
• Fluoroscence In Situ Hibridization (FISH)
• MLPA
• Chromosomal Microarray Analysis
• Single gene testing• Sanger sequencing
• MLPA and array CGH for exon deletion
• Next generation sequencing (NGS)• Panels
• Exome sequencing
• Whole genome sequencing22
CYTOGENETICS
Classical
Karyotypeanalysis
Bandingtechniques
MolecularFISH
aCGH
Diagnostic algorithm for known causes of genetic diseases
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Karyotyping&banding
FISH
diagnostic aCGH
Sangersequencing
MLPA
diagnosticaCGH
NGS panels
SNP array
PCR-
based
methods
When is chromosome analysis indicated? Problems noted during early growth/development
Stillbirths and neonatal deaths
Fertility problems
Pregnancy in women 35 years or older at the time of delivery
Family History
Strongly suggestive dysmorphic features forchromosomal anomalies
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Main indicatons of cytogenetictesting
Karyotyping
Dysmorphic childsince birth
Presence of developmental delayin a child since birth
FISH
Suspected specificchromosomal
anomaly by clinicianor researcher
Sky-FISH and M-FISH
Presence of marker chromosome of unknown origin
aCGH
Mainly in genetictyping of tumors and in idiopathic mental
retardation
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Main indicatons of cytogenetictesting accorrding to available
specimen
Availablemetaphases
Diagnosis is done Diagnosis is not done
FISH
M-FISH
SKY-FISH
aCGH
No available metaphases
Suspected specificchromosomal anomaly
by clinician or researcher
Expected or knownanomaly
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Classical cytogenetics Molecular cytogenetics
FISH aCGH
Karyotype analysisVarious dividing cells can be used:
•The most popular: peripheral blood lymphocytes
•Bone marrow cells, skin fibroblasts
•Amniocytes or trophoblast cells – in prenatal
diagnosis or in spontaneous abortions
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• 2-5 ml (1 ml in newborns) of peripheral blood is taken
• Special heparin containing tubes are needed
• Blood should be stored at 4ºC or 37 ºC (depending on lab procedures)
• Blood samples can be send by post
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\Blood
Cells
PHA
Mitogen Colcemid
Preparation
Culture Centrifugation
Cell
Pellet
Culture
medium
KCl- hypotonic
Centrifugation
Centrifugation
Cell
pelletFixation
Pipeting
Slide preparation
Drying
Slide
Staining
Giemsa solution
Cover
slide
Chromosome
documentation
Analysis
Microscope
Metaphase, chromosome spread
Karyotyping
Karyogram preparation
Banding techniques
• GTG-banding with Giemsa stain
• Q (fluorochrome: quinacrine)
• C-banding (centromeres)
• AgNor (satelites of acrocentric chromosomes)
• R-banding (reverse to GTG)
G - bands
Chromosomes are
subjected to controlled
digestion with trypsin
before staining with
Giemsa,
a DNA-binding chemical
dye.
C - bands
Is thought to
demonstrate
constitutive
heterochromatin,
mainly at the
centromeres.
Ag-NOR - banding
Is thought to demonstrate satellites of acrocentric chromosomes
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R - bands
R-banding (Reverse) to GTG banding pattern
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Q - bands
Fluorescent dye (ex. Quinacrine, DAPI) binds to AT- rich DNA
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FISH in medical genetics
• Diagnostics of submicroscopic chromosomal aberrations
• Identification of the complex chromosomal aberrations
• Identification of the additional genetic material in genome
• Identification of marker chromosomes
• Rapid prenatal diagnostics
Slide with chromosomesMolecular probeSlide with chromosomes
Denaturation of chromosomal
DNA
Double stranded DNA
In situ hybridization
Probe denaturation,
probe labelling
Direct probe detection
or
Indirect probe detection
Microscopic analysis of the
fluorescent signal on the slide
WCP 20
WCP 15
WCP 2
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Alternatives for conventional cytogenetic
analysis and FISH in prenatal diagnosis &fetal
chromosomal abnormalities detetection:
QF-PCR (qantitive fluoroscent polymerase chain
reaction)
Resolution of cytogenetic techniques
46,XX,t(4;7)(p15.2;q11.2)
M. Connor, M. Ferguson-Smith, Podstawy genetyki
medycznej, PZWL 1998
FISH: 40 -250 kbp MLPA: 40 – 60 bp
GTG: 5 – 10 Mbp aCGH: about 10 kbp
array CGH
Advantages: Whole genome screening
(depends on format) Higher resolution
Detects duplications
Limitations:
Do not detect balanced
aberration
Interpetation
Fig. CGH microarray formats
Diagnostic aCGH
Sanger Sequencing
• Gold standard for single gene testing and validation
• Limitations: must know what you are lookingfor
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MLPA
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SNP array