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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47

MLPA

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SNP array