Microdeletion Studies Using Fluorescence in Situ Hybridization

BACKGROUND:

Direct labeled probes which correspond to chromosomal regions deleted in a number of disorders can be applied to chromosomal spreads on slides prepared by routine cytogenetic techniques. Submicroscopic deletions, not visible by routine G-banding can be detected by this technique.

INDICATIONS FOR TESTING:

Testing is currently available for the following syndromes:

SYNDROME     CHROMOSOME LOCATION   PROBE/GENE LOCUS

Wolf Hirschhorn            4p16.1                                          WHS

Cri-du-Chat                     5p15.2                                         CDC

Williams                           7q11.23                                       ELN

Angelman                        15q11-q13                                  D15S10

Prader-Willi                     15q11-q13                                  SNRPN

Miller Dieker                   17p13.3                                       L1S1

Smith Magenis               17p11.2                                       SMS

DiGeorge                         22q11.2                                      TUPLE1

Steroid Sulfatase,          Xp22.3                                        STS

Kallman                           Xp22.3                                        KAL

Male Determing Factor Yp11.3                                        X/SRY

In addition we can analyze any or all individual subtelemeric regions.

SAMPLE REQUIREMENTS:

Five milliliters of whole blood collected by venipuncture into a green top sodium heparin tube. In newborns, 1-2 mls of blood may be sufficient.

SPECIMEN HANDLING:

Blood may be stored at room temperature overnight, refrigerated up to three days, but never frozen. Blood may also be mailed by overnight courier at ambient temperature. Blood drawn more that five days prior to receipt, or in the wrong tube (such as lithium heparin), is unacceptable.

PROCEDURE:

Slides are prepared in the routine manner and immediately placed at -20C until ready to use. Slides then undergo a series of dehydration and denaturation steps prior to hybridization with the probe. Hybridization generally occurs overnight in a warm humidified chamber. After a series of post washing procedures, the fluorescent signal is detected by amplification of the signal and counterstaining. The signal is then detected under a fluorescent microscope and digitized on a fluorescent imaging system.

INTERPRETATION:

Ten metaphase cells are generally counted and observed for the presence of the specific fluorescent signal. Deletions are detected by the consistent absence of the signal in one of the two homologous chromosomes.

RESULTS:

Generally, routine chromosomal analysis is performed in conjunction with FISH studies for microdeletions syndromes. The chromosome results are presented according to the international standards for chromosomal nomenclature (ISCN 2005), and a full explanation of the karyotype and clinical implications is provided. A full description of hybridization studies is provided. Any abnormal or variant results are followed by genetic counseling.

SPECIAL NOTES:

Analysis for Prader-Willi/Angelman Syndrome can be accomplished by both cytogenetic and molecular methods (see molecular section). The use of those different technologies is complementary. The decision as to which tests are appropriate for any sample is made on a case by case basis. The usual procedure is to perform cytogenetic and FISH studies first and molecular studies if these are negative. Therefore both green and purple top tubes should be submitted for any diagnosis.

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