The Principles of Clinical Cytogenetics - Extra Materials - Springer

Fluorescence In Situ Hybridization 467
performed on the blastomeres or polar bodies. Embryos shown to be free of the genetic disease under
investigation are transferred to the uterus. Multicolor FISH can be used to diagnose numerical and
certain structural abnormalities of chromosomes in the embryo, and this methodology has been
adopted by most PGD centers worldwide as the method of choice for sex determination and for
diagnosis of aneuploidy (43). As with prenatal diagnosis, the common probes used for ploidy assessment
are for chromosomes 13, 18, 21, X, and Y. FISH with subtelomeric probes is useful for PGD of
translocations when one of the parents is a known carrier.
Although FISH is the most widely used method for PGD for some genetic diagnoses, there are several
limitations with this technology (43). FISH is generally limited to diagnosis at the chromosome level
rather than the single-gene level. Therefore, other methods are needed for single-gene defects such as
cystic fibrosis. Also, misdiagnosis (both false positive and false negative) is relatively common and has
been reported in as many as 21% of single-cell assessments (44). In addition, analysis is often limited to
the study of five chromosomes because of the restricted number of fluorochromes and the need to eliminate
technical artifacts (overlapping signals) in a single cell. However, for couples with a high risk of
having a child with a genetic disease, PGD using FISH is valuable for assessing embryo sex and chromosome
number so that selective abortion and/or the birth of an affected child can be avoided.
Sex Chromosome Abnormalities
Certain sex chromosome abnormalities, such as the XX male (see Chapter 10), cannot be satisfactorily
diagnosed with cytogenetics alone. Because most such patients are SRY positive, FISH analysis
with probes for the X chromosome and SRY is typically necessary to confirm the diagnosis (see Fig 8).
FISH Applications for Studies of Acquired Chromosomal Aberrations
One major area that has been advanced greatly by FISH is the study of chromosomal abnormalities
associated with cancer (see Chapters 15 and 16). Probes have been developed for the majority of
recurrent aberrations found in hematologic malignancies, and the National Cancer Institute (NCI)
has undertaken an endeavor to produce resources for the genetic study of solid tumors. Cancer-specific
FISH probes and their characteristics are presented in Table 5. Several of these diseases and appropriate
probes are discussed in detail below.
Acute Myelogenous Leukemia
Approximately 40–60% of acute myelogenous leukemia (AML) patients exhibit genetic aberrations
that are easily detected by FISH, and in 2001, the World Health Organization (WHO) established
an AML classification system that was based on recurrent genetic abnormalities (45) (see also
Chapter 15). For each category, classical cytogenetics identifies the majority of aberrations; however,
FISH can be used to detect cryptic abnormalities and variant rearrangements and to monitor
disease states during and following treatment.
The t(8;21) juxtaposes the AML1 gene on chromosome 21 and the ETO gene on chromosome 8. A
dual-color, dual-fusion (DCDF) probe has been developed to detect the fusion products on the derivative
8 and the derivative 21 chromosomes (see Fig. 9). Similarly, a DCDF probe can be used for
AML with t(15;17) in which there is a juxtaposition of the retinoic acid receptor-α (RARα) gene at
17q12 and the PML (promyelocytic leukemia) gene at 15q22. FISH with the dual-fusion probes provides
a definitive diagnostic test and a sensitive assay for minimal residual disease assessment. Rapid
FISH diagnosis (8–48 hours) of the PML/RARα fusion is of utmost importance, so that patients can
begin appropriate therapy with all-trans retinoic acid (ATRA). In addition, FISH studies allow for the
differentiation of promyelocytic leukemia with t(15;17), as opposed to a variant such as t(11;17).
This is clinically significant, because patients with variant translocations do not respond to ATRA
treatment. The t(11;17) and other RARα variants can be identified with a RARα break-apart probe.
Acute myelogenous leukemia with inv(16)(p13q22) or t(16;16)(p13;q22) results from the fusion
of the core-binding factor-β (CBFβ) gene at 16q22 to the muscle myosin heavy chain (MYH11) at