Happie Haema

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FLUORESCENT IN SITU

HYBRIDIZATION (F.I.S.H.)

AS A PRE-NATALDIAGNOSTIC TOOL.

BY

ALIU U. HAPPINESS

DISCUSSANT: BIVAN MURNA

CO-ORDINATOR: A. J. WHYTE.

JANUARY, 2013.


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OUTLINE.

INTRODUCTION

HISTORY.

IMPORTANCE OF FLOURESCENCE IN SITU

HYBRIDIZATION (F.I.S.H.)

THE USE OF F.I.S.H IN PRE-NATAL DIAGNOSIS.

LIMITATIONS OF F.I.S.H.

CONCLUSION.

RECOMMENDATION.

REFERENCES.


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INTRODUCTION.

Fluorescence in situ hybridization

(F.I.S.H.), is the assay of choice for

localization of specific nucleic acid

sequences in native context.

Fish is a DNA mapping technique in which

a DNA probe labeled with a marker

molecule is hybridized to chromosomes ona slide and visualized using a fluorescence

microscope.


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HISTORY.

In the 1960s, researchers Joseph Gall and

Mary Lou realized that molecular

hybridization could be used to identify the

position of DNA sequences in situ (i.e. in

their natural positions within a

chromosome).

In 1969, the two scientists published alandmark paper demonstrating that

radioactive copies of DNA sequence could


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Historycontd.

be used to detect complementary DNA

sequences in the nucleus of a frog egg.

Soon after Gall & Lous work, fluorescent

labels quickly replaced radioactive labels

in hybridization probes because of their

greater safety, stability and ease of

detection.

Today, most in situ hybridization is done

using FISH procedures.


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BASIC PRINCIPLES OF

F.I.S.H. The basic elements of F.I.S.H. are a DNA

probe and a target sequence.

Before hybridization, the DNA probes are

labeled with a fluorophore.

Combining the denatured probe and target

allows the annealing of complementary

DNA sequences.


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THE HUMAN CHROMOSOME.

Normalfinding:

46,XY

Micro-deletionshould be

confirmed by

the FISH

analysis


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IMPORTANCE OF F.I.S.H. IN

PRE-NATAL DIAGNOSIS. Pre-natal diagnosis involves testing for

disease conditions in a fetus before its

birth, with the aim of detecting birth

defects, chromosome abnormalities andgenetic diseases.

This may sound as a total waste of time,

however, women over the age of 35 andthose with high blood pressure, diabetes,

asthma, epilepsy as well as those with


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Contd.

family histories or ethnic backgrounds prone

to genetic disorders, or whose partners

have these are likely to give birth to babies

with some birth defect.

Hence, the need for pre-natal diagnosis to

give the parents the chance to prepare

psychologically, socially, financially andmedically for a baby with a health problem/

disability, or for the likelihood of a stillbirth.


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INDICATIONS FOR PRE-

NATAL TESTING. History of multiple miscarriage.

When parents have children with known

genetic disorders.

When prospective mother is more than 35

years of age.

When either parents have produced achild with chromosome abnormality.

When either parents is a mosaic or carrier

of a chromosome anomaly.


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Case 1: DiGeorge syndrome

hypertelorism

micromandibulalow set dysplastic

ear

antimongoloid

slant of eyelids


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Microdeletion confirmed (loss of

one red signal)

Red signal

TUPLE1 (HIRA)

locus

Green signal

ARSA locus

(control probe)

Deleted chromosome

red signal absent

normal

chromosome

red signal on

HIRA locus is

present

Microdeletion 22q11.2 is associated with

DiGeorge syndrome.


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Case 2

Phenotypic features and inborn defects

are typical for Williams-Beuren syndrome

This syndrome is caused by micro-deletion

of the long arm of the chromosome 7 (sub-

band 7q11.23).

It presents with inborn cardiac defects as

well as mental retardation.


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Case 2 (boy, 2 years)

irides

stellataehypertelorism

open mouth,

thick lip

abnormal teeth

elfin face

low set ears


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OTHER APPLICATIONS OF

F.I.S.H. Gene mapping.

Chromosome identification.

Aneuploidy detection. Total chromosome analysis.

Unique sequence DNA detection.

Gene amplification analysis. Micro-deletion syndrome analysis.


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PROS OF F.I.S.H.

Advantage: less labor-intensive method for

confirming the presence of a DNA

segment within an entire genome than

other conventional methods like Southernblotting.

F.I.S.H. assays have high levels of

reliability, reproducibility and accuracywhen compared with other pre-natal

diagnostic techniques.


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LIMITATIONS OF F.I.S.H.

Fish does not look at the actual structure

of the chromosome analyzed, it only tells

how many copies of a particular

chromosome are present.

Probe availability must be cleared with the

laboratory before specimen is collected,

because probes often have limitedavailability.


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CONCLUSION

FISH - a process which vividlypaints chromosomes or portions of

chromosomes with fluorescent

molecules. FISH results provide couples with a

means to make rational andinformed decisions concerning the

pregnancy in cases where

anomalies are detected.


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RECOMMENDATION.

Pre-natal diagnosis can detectchromosome abnormalities

associated with unexplained mental

retardation and miscarriages.

Therefore, it is recommended that

Teaching hospitals and otherclassic health institutions should

request F.I.S.H. for pregnant

mothers at risk.


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REFERENCES.

Dobzhansky T. (2012), Genetics and the

origin of species. Columbia University

Press, New York 20: 10-15.

Dolan S.M. (2011), Prenatal Genetic

Testing. Pediatr.Ann 38: 426-430.

Kottler M. (2009), Preconception and the

counting of the Human Chromosomes.

Bull Histo Med48: 465-502.


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Contd

Macdonald F. (2008), Practice of Pre-nataldiagnosis in the UK. Clinc Risk6: 14:

218-221.

Painter T.S. (2012), A new method for the

study of chromosome re-arrangements

and the plotting of chromosome maps.

Science 78: 585-586.

Stefansdottir V. (2010), Acceptance of 1st

trimester screening for chromosomal

anomalies. Obstet Gynaecol89: 931-

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