FLUORESCENT IN SITU HYBRIDIZATION (F.I.S.H.) AS A PRE-NATAL DIAGNOSTIC TOOL.

BY ALIU U. HAPPINESS DISCUSSANT: BIVAN MURNA CO-ORDINATOR: A. J. WHYTE. JANUARY, 2013.

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.

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 on a slide and visualized using a fluorescence microscope.

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 a landmark paper demonstrating that radioactive copies of DNA sequence could

History contd.
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.

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.

THE HUMAN CHROMOSOME.

Normal finding:
46,XY

Micro-deletion should be confirmed by the FISH analysis

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 and genetic diseases. This may sound as a total waste of time, however, women over the age of 35 and those with high blood pressure, diabetes, asthma, epilepsy as well as those with

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 and medically for a baby with a health problem/ disability, or for the likelihood of a stillbirth.

INDICATIONS FOR PRENATAL 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 a child with chromosome abnormality. When either parents is a mosaic or carrier of a chromosome anomaly.

Case 1: DiGeorge syndrome

antimongoloid slant of eyelids hypertelorism

low set dysplastic ear

micromandibula

Microdeletion confirmed (loss of one red signal)

Deleted chromosome red signal absent

Red signal TUPLE1 (HIRA) locus Green signal ARSA locus (control probe)
normal chromosome red signal on HIRA locus is present

Microdeletion 22q11.2 is associated with

DiGeorge syndrome.

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 (subband 7q11.23). It presents with inborn cardiac defects as well as mental retardation.

Case 2 (boy, 2 years)

hypertelorism
irides stellatae

low set ears abnormal teeth

open mouth, thick lip

elfin face

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.

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 Southern blotting. F.I.S.H. assays have high levels of reliability, reproducibility and accuracy when compared with other pre-natal diagnostic techniques.

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 limited availability.

CONCLUSION
FISH - a process which vividly paints chromosomes or portions of chromosomes with fluorescent molecules. FISH results provide couples with a means to make rational and informed decisions concerning the pregnancy in cases where anomalies are detected.

RECOMMENDATION.
Pre-natal diagnosis can detect chromosome abnormalities associated with unexplained mental retardation and miscarriages. Therefore, it is recommended that Teaching hospitals and other classic health institutions should request F.I.S.H. for pregnant mothers at risk.

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 Med 48: 465-502.

Contd
Macdonald F. (2008), Practice of Pre-natal diagnosis in the UK. Clinc Risk 6: 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 Gynaecol 89: 931938.