Fig. 17.1
Schematic representation of the basic steps of the FISH procedure. Both the probe and chromosomal target are heat-denatured. Probe sequences hybridize to the complementary target sequences, and nonspecific binding is eliminated via stringent washing. The probe hybridization is detected with fluorescence microscopy
Probe Types
Given the abundance of sequence data available from the Human Genome Project, probes amenable for FISH procedures may be produced for the study of almost any human chromosomal site. However, the majority of probes used for clinical purposes are commercially manufactured and sold as analyte-specific reagents (ASRs) that must be validated by each laboratory. Most FISH probes fall into one of three categories: repetitive sequence, whole chromosome, or unique sequence. The most widely used repetitive sequence probes are for the alpha satellite sequences located at the centromeres of human chromosomes. Alpha satellite DNA is composed of tandemly repeated monomers, thus the sequences targeted by the probes are present in several hundreds or thousands of copies, producing strong signals. Each chromosome’s alpha satellite sequence (with the exception of chromosomes 13 and 21 and chromosomes 14 and 22) is sufficiently divergent to allow for the development of centromere-specific probes. These probes are particularly useful for detection of aneuploidy in both metaphase and interphase cells. In addition, alpha satellite probes are useful for the detection of acquired monosomy or trisomy in malignancies, such as trisomy 12 in chronic lymphocytic leukemia, or monosomy 7 or trisomy 8 in myeloid disorders (see Chap. 15). Other types of repetitive sequences for which probes have been developed include the beta satellite sequences (located in the short arms of the acrocentric chromosomes), “classical” satellite sequences (found at various locations including the heterochromatic region of the Y chromosome), and telomeric repeat sequences (TTAGGG) that mark the physical ends of each human chromosome. These latter probes are not as routinely used in the clinical setting but are valuable for the study of structural aberrations.
Whole chromosome probes (WCP), also known as chromosome libraries or chromosome “painting” probes, are composed of unique and moderately repetitive sequences from an entire chromosome or chromosomal region. The generation of this type of probe requires that DNA from a particular chromosome be isolated from the rest of the genome. This may be accomplished using flow sorting, somatic cell hybrids containing a single human chromosome or area of a chromosome, or microdissected chromosomes and subsequent amplification of the dissected DNA sequences via the polymerase chain reaction (PCR) [7]. WCPs are commercially available for each human chromosome and are most frequently used for the study of structural aberrations. For example, WCPs may be used to identify the chromosomal origin of additional unknown material of derivative chromosomes and also to confirm the cytogenetic interpretation of translocations (Fig. 17.2).
Fig. 17.2
Characterization of a structurally abnormal chromosome 7 in a patient with an unbalanced translocation. A chromosome 17 library (“painting” probe) was applied to peripheral blood metaphase cells. Both normal chromosomes 17 hybridized entirely, and the unidentifiable material attached to the short arm chromosome 7 (arrow) is also painted. The patient therefore has three copies of sequences from chromosome 17
The third and most widely used type of probe is for unique sequence DNA. These probes are generated from regions of the genome that are either cloned into various vectors (e.g., cosmids, yeast artificial chromosomes [YACs], bacterial artificial chromosomes [BACs]) or are made by PCR using sequence-specific primers. Some probes include extraneous repetitive sequences, and Cot-1 DNA must be added to the hybridization mixture to block nonspecific binding so that only the unique sequences are visualized. Other probes, termed single-copy probes, are designed and developed based on genomic sequences that are devoid of repetitive sequences [8, 9]. Unique sequence probes, which range in size from approximately 1 kilobase (Kb) to >1 megabase (Mb), may be used to examine a particular area for copy number or location. For example, probes developed to span a translocation breakpoint, such as a probe for the 5′ and 3′ regions of the MLL gene, allow for detection of cryptic translocations involving this important cancer locus. These probes are also useful for delineating chromosomal breakpoints and for allowing the visualization of copy number changes detected by genomic microarray hybridization.
Labeling and Detection
The majority of probes that are used in the clinical cytogenetics laboratory are directly labeled and commercially available. However, probes can be indirectly labeled via incorporation of a hapten (such as biotin or digoxigenin) into the DNA via nick translation or PCR. The haptens are attached to the probe nucleotides and are detected by a secondary reaction using a fluorescently labeled antibody. The most common indirect systems involve biotin-streptavidin or digoxigenin-antidigoxigenin. Fluorochromes, such as rhodamine, Texas Red, or fluorescein, may be conjugated to the streptavidin or antidigoxigenin and detected upon excitation with a fluorescence microscope. Alternatively, directly labeled probes, with the fluorochrome attached to the probe nucleotides, require no secondary detection and may be directly visualized after fluorescent excitation.
Specimen Types
FISH can be applied to a variety of specimen types depending upon the study of interest. Metaphase preparations from cultured cells (amniocytes, chorionic villous cells, lymphocytes, cells from bone marrow aspirates or solid tumors, fibroblasts) that are routinely utilized for cytogenetic analysis are optimal preparations for FISH studies as well. FISH on metaphase cells is considered the “gold standard” because the chromosomes and the exact position of the signals can be visualized directly. However, one major advantage of FISH is that it can also be performed on interphase cells. Interphase nuclei assessment from uncultured preparations allows for rapid screening for prenatal diagnosis (amniocytes for ploidy analysis), for newborn studies (peripheral blood smears for ploidy analysis), or for cancer studies (bone marrow aspirate smear or direct harvest for translocation or copy number analysis). In addition to uncultured cells, interphase analysis may also be performed on slides prepared for routine chromosome analysis, paraffin-embedded tissue block sections, disaggregated cells from paraffin blocks, and touch preparations of cells from lymph nodes or solid tumors. For cases in which metaphase chromosomes are limited, of poor quality, or unavailable, FISH provides a means for assessment when the routine chromosome analysis would otherwise be considered a failure. Analysis of interphase cells also allows for an increased number of cells to be assessed. Given that interphase studies cannot be verified by visualization on in situ chromosomes, interpretation may be compromised by hybridization inefficiency, and quality assurance is of the utmost importance.
Clinical Applications
Constitutional FISH Studies
One major advantage of FISH is its ability to detect and characterize chromosomal abnormalities that are not routinely delineated with standard banding studies. This technology allows for the detection of subtle deletions or duplications, identification of marker chromosomes, and characterization of other chromosomal rearrangements. In addition, FISH is used to visualize aberrations that are detected by copy number microarray analyses and to assess parental samples for the copy number change and/or definition of a balanced rearrangement.
Microdeletions and Microduplications
Microaberrations or contiguous gene syndromes are caused by the deletion or duplication of genetic material, usually involving multiple contiguous genes on a chromosome (Table 17.1). Breaks often occur at consistent locations and are mediated by low copy repeats (LCRs) that permit nonallelic homologous recombination. These contiguous gene syndromes, which often involve deletions or duplications that are 2 Mb or less in size, cannot be identified with routine chromosome studies. Therefore, FISH analysis provides a definitive diagnostic test for these disorders.
Table 17.1
Microdeletion syndromes
Syndrome | Deletion | Probea | Phenotype |
|---|---|---|---|
Angelman | 15q11.2–15q13 | SNRPN | Severe mental retardation; hypotonia; ataxia; lack of speech; hypopigmentation; seizures; inappropriate laughter; and dysmorphic features |
D15S10 | |||
DiGeorge | 22q11.2 | D22S75 | Dysmorphic features; congenital heart disease; absence of thymus; growth failure; cognitive deficits |
HIRA | |||
Miller-Dieker | 17p13.3 | PAFAH1B1 | Severe mental retardation; lissencephaly; dysmorphic facial features |
Prader-Willi | 15q11.2–15q13 | SNRPN | Mental retardation; hypotonia; feeding difficulty; genital hyperplasia; obesity; hyperphagia; and dysmorphic features |
Smith Magenis | 17p11.2 | SHMT1 | Mental retardation; speech delay; bizarre behavior; peripheral neuropathy; and dysmorphic facial features |
TOP3A | |||
FLI1 | |||
LLGL1 | |||
Velo-Cardio Facial | 22q11.2 | HIRA | Delayed development; pharyngeal deficiency; abnormal facies; palatal defects; and congenital heart defects |
Williams | 7q11.2 | ELN | Mental retardation; hypercalcemia; elfin facies; gregarious personality; and congenital heart disease |
Angelman and Prader-Willi syndromes, each of which occurs in approximately 1/10,000 individuals, involve the loss of expression of the maternal or paternal genes, respectively, in 15q11.2–15q13. Approximately, 70% of cases are due to a deletion (Fig. 17.3a). Other causes include uniparental disomy (UPD), imprinting mutations (see Chap. 20), and, for Angelman syndrome, mutations of the UBE3A gene. The deletions involve approximately 2–5 Mb of DNA and may be detected by FISH with a probe for the SNRPN gene or other genes in the region. Approximately 90% of the deletions occur at the same distal breakpoint and involve one of two proximal breakpoints [10, 11]. The reciprocal product of the unequal crossing-over event, resulting in duplications of 15q11-q13, has been associated with autism (Fig. 17.3b).
Fig. 17.3
Example of FISH to a single-copy target using a cosmid (SNRPN) to the Prader-Willi “critical region” localized to 15q11-13. (a) A metaphase in which one normal chromosome 15 has three hybridization signals from a centromeric control probe (green), a distal control probe (red), and a probe to the critical region (red). The other chromosome 15 (arrow) revealed hybridization signals only for the two control probes. Thus, this chromosome was deleted for the critical region, and this patient was diagnosed with Prader-Willi syndrome. Chromosomes were counterstained blue with DAPI. (b) In this partial metaphase, a SNRPN probe and a control probe (both yellow) were utilized (current standards and guidelines require the use of different fluorochromes; see Chap. 6). Chromosomes were counterstained orange with propidium iodide. The arrow indicates the chromosome 15 with a duplicated SNRPN signal. This patient was referred for a diagnosis of autism. See text
Several disorders involving unequal crossing-over mediated by LCRs in the short arm of chromosome 17 are routinely studied by FISH analysis [12]. Miller-Dieker syndrome involves the loss of ∼2 Mb of DNA in 17p13.3 including the PAFAH1B1 (formerly LIS1 or lissencephaly 1) gene and other gene(s) responsible for the dysmorphic features [13]. FISH with a probe for the PAFAH1B1 gene allows for the detection of the Miller-Dieker syndrome deletion and may also be useful for a proportion of cases with isolated lissencephaly. Another LCR-mediated mechanism results in a deletion of chromosome 17 at band p11.2 causing Smith-Magenis syndrome or a duplication of this region resulting in dup(17)(p11.2p11.2) syndrome, both of which can be diagnosed using FISH with probes for the critical region [14]. Similarly, interphase FISH with a probe for a 1.4 Mb area of 17p12 may be used to detect the duplication associated with Charcot-Marie-Tooth disease 1A. This same region is deleted in patients with hereditary neuropathy with liability to pressure palsies (HNPP).
Microdeletions of 22q11.2, resulting in velocardiofacial (VCF) or DiGeorge syndrome, are seen in about 1/2,000–1/3,000 individuals. Because of the relatively high frequency of this syndrome and its association with congenital heart disease, fetuses and newborns with a heart defect routinely undergo FISH testing for a 22q deletion. This syndrome, in contrast to other microdeletion syndromes, is inherited in about 10% of the cases. Therefore, FISH studies are recommended for parents of an affected individual. Most patients with VCF or DiGeorge syndrome have an LCR-mediated deletion of ∼3 Mb; some patients have a smaller ∼1.5 Mb deletion that is caused by an internal LCR [15]. Mutations in the TBX1 gene have been correlated with the abnormal phenotype, and this gene is a candidate for the psychiatric disease associated with VCF and DiGeorge syndrome [16, 17]. The reciprocal syndrome caused by duplication of 22q11.2 is associated with dysmorphic features, growth failure, cognitive deficits, hearing loss, and velopharyngeal insufficiency [18]. Both the microdeletion and the microduplication of 22q11.2 are easily detected by FISH with a probe for the HIRA gene or a probe for the DNA segment D22S75.
Williams syndrome involves the loss of genes in the long arm of chromosome 7 at band q11.23. The deletion has two major breakpoints that are mediated by LCRs. The deletion usually cannot be detected by G-banding, but can routinely be detected by FISH with a probe for the elastin (ELN) gene. Phenotypic features seen in this syndrome elegantly demonstrate the definition of a contiguous gene syndrome, as Williams syndrome involves both the central nervous system and connective tissue abnormalities. Abnormalities include mental retardation, infantile hypercalcemia, elfin facies, dysmorphic facial features, a gregarious personality, premature aging of the skin, and a congenital heart disease (supravalvular aortic stenosis) [19, 20].
Cryptic Subtelomeric Rearrangements
It is generally accepted that even with “high-resolution” chromosome analysis, alterations of chromosomal material of less than 2–4 Mb cannot be detected. In particular, due to the small size of aberration and exchange of similarly banded (G-negative) material, visualization of abnormalities in the telomeric regions is difficult. Given that these regions are gene-rich, they have particular relevance for clinical studies.
Aberrations of the subtelomeric regions have been documented in a significant percentage of patients with idiopathic mental retardation with an overall frequency of approximately 5% (range of 0–13.3%) [21, 22]. The majority of subtelomeric studies have been performed using FISH, and, in general, these studies have confirmed the efficacy of using subtelomeric probes for the assessment of individuals with mental retardation, with some cautionary notes. Not all studies used the same set of probes, and depending on the location of some probes, there was a high likelihood of detection of polymorphisms with no clinical significance, skewing the detection rates reported. Polymorphisms resulting in deletions, duplications, and other rearrangements of subtelomeric regions have been confirmed with family studies. Of note, small terminal deletions detected cytogenetically are also commonly detected by subtelomeric FISH probes. These areas of involvement include 1p, 1q, 2q, 8p, 10q, and 22q [23–26].
While FISH has historically been the method of choice to study the subtelomeric areas, chromosomal microarray analysis that targets the entire genome, including the subtelomeric areas, has largely replaced FISH testing for patients with mental retardation/development delay and autism (see Chap. 18). Subtelomeric FISH probes are still valuable, however, as an aid.
Duplications and Marker Chromosomes
Characterization of de novo duplications and marker chromosomes has valuable implications with respect to phenotype/karyotype correlation. Approximately 70% of chromosomal duplications are intrachromosomal (Fig. 17.4), while 30% involve a nonhomologous chromosome [27]. Although chromosomal microarray studies are the optimal method to determine the genomic content of duplicated segments, FISH with chromosomal paints probes, locus-specific probes, and/or M-FISH (see section “Specialized and Evolving FISH Technologies” later in this chapter) may also be used to identify the chromosomal origin of extra material.
Fig. 17.4
Partial metaphase spread from a patient with a duplication involving chromosome 11. A BAC localized to chromosome 11p15.5 produced one signal on the normal chromosome 11 and a double signal on the duplicated chromosome 11 (arrow). The duplication in the short arm of chromosome 11 was detected in a newborn that was large for gestational age. The infant also had an omphalocele and was diagnosed with Beckwith-Wiedemann syndrome
Chromosomes that are unidentifiable by routine banding are termed “markers” (see Chaps. 3 and Chaps. 8). Marker chromosomes represent a heterogeneous group and are typically extra structurally abnormal chromosomes (ESACs). The most common types of markers, for which clinical phenotypes have been defined, may be fully characterized using FISH (Table 17.2). Other types of markers may be partially defined by FISH, and the impact of these chromosomes on the clinical phenotype often cannot be reliably predicted. Many marker chromosomes are present in mosaic form and cannot be characterized by use of chromosomal microarray analysis.
Table 17.2
Marker chromosome assessment
Type of marker | FISH probe result | Associated syndrome/phenotype (estimated risk for abnormality)a |
|---|---|---|
ESAC | Pan-centromeric, no alpha satellite | High risk for abnormality; phenotype dependent upon euchromatin present |
Bisatellited/monocentric | Alpha satellite +: 13/21, 14/22, 15 | General risk for bisatellited = 11% |
idic(15) | 95% – MR | |
SNRPN – positive | ∼0% risk | |
SNRPN – negative | 5% – MR (Usually due to UPD) | |
idic(22) | ATP6V1E1 – present | Cat eye syndrome |
Monosatellited | Alpha satellite +: 13/21, 14/22 | No general risk, dependent on whether euchromatic material present |
Nonsatellited metacentric | Alpha satellite present | General risk for nonsatellited = 11% |
Alpha satellite present for 8, 9, 12, or 18 centromere | If metacentric, risk for MR = ∼100% | |
Sex chromosome | DXZ1 (X centromere) + | |
XIST – positive | Turner syndrome only >95% | |
XIST – negative | Majority – MR | |
DYZ3 (Y centromere) | ||
SRY – positive | Male phenotype | |
SRY – negative | Female phenotype |
Identification of chromosomal origin can be accomplished by using M-FISH (see later in this chapter), or utilizing individual chromosomal libraries or alpha satellite DNA probes. Characteristics, such as shape and size of the marker chromosome, determine what probes are best for FISH studies. If the marker is metacentric, it is likely to be an isochromosome (see Chap. 3) and should be studied with alpha satellite probes from chromosomes 8, 9, 12, and 18, as these are the most likely isochromosomes to be present. These are all associated with an abnormal phenotype. If the marker is satellited (or bisatellited), DNA probes from the centromeres of chromosomes 13/21, 14/22, and 15 should be used. Once the origin is determined, that information, along with the structure, dictates the additional studies to be done. For example, regardless of its origin, a monocentric, bisatellited chromosome is often not associated with an abnormal phenotype, whereas a monocentric, monosatellited chromosome may be. If a satellited marker is derived from a chromosome 15, SNRPN status should be determined (Fig. 17.5). If SNRPN is present, the karyotype would be associated with an abnormal phenotype [28].
Fig. 17.5
A dicentric chromosome hybridized with dual-color chromosome 15 probes, including both an alpha satellite DNA probe (green) and a single-copy SNRPN probe (red). Signals from both probes are present on the normal chromosomes 15. The marker chromosome (arrow) has two alpha satellite DNA signals, confirming that it is dicentric. In addition, the marker contains two copies of the SNRPN probe. A control probe for the distal long arm was also included; signals are only present on the normal chromosomes 15 and not on the marker chromosome. This abnormality was ascertained in a 6-year-old female with hypotonia, behavior and learning problems, and autism
Sex chromosome markers are usually found in individuals who have 46 chromosomes, with only one normal X and a marker chromosome in place of a second sex chromosome. These abnormal chromosomes should be initially studied with X and Y alpha satellite probes. If the marker originates from an X chromosome, it should be studied with a probe for the XIST gene (the gene responsible for initiation of X inactivation; see Chap. 10). If XIST is absent, the phenotype will likely be associated with mental retardation/developmental delay [29]. If the marker originates from a Y chromosome, FISH with a probe for SRY should be performed to better understand the marker’s effect on phenotype. Patients with marker chromosomes that are Y-derived are at risk for gonadoblastoma; thus, it is of significant importance to document the origin of sex chromosome markers.
The last category of markers involves ring or marker chromosomes that cannot be placed into any of the other groups. M-FISH or FISH along with each alpha satellite probe is useful for determining the chromosomal origin of such markers. However, this information does not usually allow for specific clinical risk estimations for genetic counseling (see Chap. 21).
Follow-Up Studies for Copy Number Aberrations Detected by Microarray Analysis
Microarray analysis, using comparative genomic hybridization or single nucleotide polymorphism platforms, has proven to be the most sensitive and highest resolution assessment of copy number changes in the genome, detecting aberrations in approximately 20% of individuals referred for developmental delay [30] (see Chap. 18). This genomic analysis can detect gains and losses of chromosomal material but cannot identify the mechanism underlying the change. Thus, while microarrays offer high-resolution analysis, further studies are often necessary to assist with the interpretation of the result. FISH with a probe or probes contained within the region designated on the microarray can be used to evaluate members of the proband’s family for the presence of balanced rearrangements or to detect familial copy number changes that are likely of no clinical significance.
Prenatal Studies
FISH has been widely used for the detection and analysis of prenatal chromosomal abnormalities (see Chap. 12). One major advantage of FISH technology is the ability to study uncultured material to produce a rapid result. In addition, FISH is useful to characterize or detect subtle abnormalities not delineated by routine banding (e.g., deletions, markers, or duplications).
Ploidy Analysis
The vast majority of abnormalities detected prenatally are aneuploidies, involving chromosomes 13, 18, 21, or the sex chromosomes. FISH provides rapid ploidy assessment of these chromosomes by utilizing probes on uncultured interphase cells from amniotic fluid or chorionic villi [31–39] (Table 17.3). In most cases, five probes are used and applied to two different slides (or two different sections of a single slide). α-satellite DNA for the X chromosome and chromosome 18 is used together with a classical satellite probe for the Y chromosome, using three different probe colors. The other mix consists of single-copy probes for both chromosomes 13 and 21, using two different colors. These studies will ascertain numerical abnormalities for these chromosomes (Fig. 17.6) and will also detect triploidy.
Table 17.3
Prenatal ploidy analysis
Study | No. | False (+) | False (−) | Uninformative |
|---|---|---|---|---|
Ward et al. (1993) [31] | 4,500 | .1% | .2% | 6.1% |
Mercier et al. (1995) [32] | 630 | 0 | (1).2% | |
Bryndorf et al. (1997) [33] | 2,000 | 0 | 0 | 7% |
Jalal et al. (1998) [34] | 508 | 0 | 0 | |
Eiben et al. (1999) [35] | >3,000 | 0 | 0 | |
Weremowicz et al. (2001) [36] | 911 | (1).1% | (5).5% | 3.0% |
Tepperberg et al. (2001) [37] | 5,197 | (1).003% | (7).024% | |
Sawa et al. (2001) [38] | 2,639 | 0 | 0 | 6.0% |
Witters et al. (2002) [39] | 5,049 | 0 | 0 | 0.26% |
Fig. 17.6
Prenatal ploidy assessment utilizing Abbott Molecular AneuVysionTM analysis of uncultured amniotic fluid cells using unique copy probes for the long arms of chromosomes 13, 18, 21, X, and Y. The results in these interphase cells are consistent with a XY fetus with trisomy 21. Left: probes for chromosomes 13 (2 green signals) and 21 (3 orange signals). Right: probes for chromosomes 18 (2 aqua signals), X (green signal), and Y (orange signal). Nuclei are counterstained blue with DAPI
While FISH assessment on uncultured cells can provide answers within 24 h of obtaining a sample, these studies are limited in that only aneuploidies for a select number of chromosomes (13, 18, 21, X, and Y) can be detected. In a 5-year collaborative study, a total of 146,128 amniocenteses were performed revealing a total of 4,163 abnormalities; however, only 69.4% of these would have been detected using interphase analysis of uncultured amniotic fluid cells [40]. A detection rate (65–70%) has been proposed in a position statement by the American College of Medical Genetics (ACMG)/American Society of Human Genetics (ASHG). The statement indicates that the sensitivity would increase to 80% with increasing age because of the association of increased age and nondisjunction.
Overall, prenatal FISH technology has been found to be effective, sensitive, and specific. Tepperberg et al. reported on a 2-year multicenter retrospective analysis and review of literature of the AneuVysion assay (Abbott Molecular) [37]. Of the 29,039 studies able to be documented, there was only one false-positive (0.003%) and 7 false-negative (0.024%) results. The results suggested that this was an effective test for aneuploidy of the testable chromosomes in cases of advanced maternal age or pregnancies indicated to be at increased risk due to maternal screening results or ultrasound findings. As this test is an adjunct test to standard cytogenetic analysis, the position statement by the ACMG/ASHG states that decisions to act on laboratory test information should be supported by two of three possible pieces of information, i.e., (1) FISH results, (2) routine chromosome analysis, and (3) clinical information (e.g., ultrasound examination).
Although much less common, these probes are also used with chorionic villus samples (CVS), in vitro fertilization (IVF) specimens, and fetal cells found in maternal blood. They can also be utilized to detect aneuploidy in paraffin-embedded specimens from pregnancy losses.
Preimplantation/Embryo Studies
Preimplantation genetic diagnosis (PGD) is the early diagnosis of genetic disorders, prior to the onset of pregnancy. Embryos or oöcytes are biopsied during culture in vitro and genetic analysis is 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 may 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 [41–43]. Some test centers use only five probes (for chromosomes 13, 18, 21, X, and Y) for ploidy assessment, but most centers increase accuracy by using 12–24 probes. For translocation carriers, FISH with subtelomeric probes is useful for detecting unbalanced zygotes.
Although FISH is the most widely used method for PGD for some genetic diagnoses, there are several limitations with this technology [41]. 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 due to the restricted number of fluorochromes and the need to eliminate technical artifact (overlapping signals) in a single cell. Even with these limitations, for couples with a high risk of having a child with a genetic disease, PGD using FISH is very 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 Chap. 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 (Fig. 17.7). For patients with a 45,X karyotype, FISH studies are recommended to determine if there is hidden mosaicism for Y-chromosomal material that could predispose the patient to gonadoblastoma [45].
Fig. 17.7
Metaphases from an XX sex-reversed male were hybridized with probes for the X centromere (green) and a probe for the SRY gene (red). Results demonstrated a cryptic translocation in which SRY was present on the short arm of one X chromosome. Chromosomes were counterstained blue with DAPI
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 Chaps. 15 and Chaps. 16). Probes have been developed for the majority of recurrent translocations found in hematologic malignancies, and there are many probes for the genetic study of solid tumors. Cancer-specific FISH probes and their characteristics are presented in Table 17.4. Several of these diseases and appropriate probes are discussed in detail as follows.
Table 17.4
FISH for hematologic malignancies
Chromosomal aberrationa | Chromosome – gene(s) involved | Disease associationb | Probe type(s)c |
|---|---|---|---|
t(9;22)(q34;q11.2) | 9 – ABL1 | CML, ALL, AML | DCDF, DCSF, DCES, FCDF |
22 – BCR | |||
t(15;17)(q22;q21.1) | 15 – PML | AML | DCDF, DCSF, BAP |
17 – RARA | |||
t(*;11)(*.*; q23) | 11 – MLL | ALL, AML | BAP |
t(8;21)(q22;q22) | 8 – RUNX1T1 | AML | DCDF |
21 – RUNX1 | |||
inv(16)(p13q22) or t(16;16)(p13;q22) | 16q22 – CBFB | AML | BAP |
t(12;21)(p13;q22) | 12 – ETV6 | ALL | DCES |
21 – RUNX1 | |||
Trisomy 8 | 8 – 8cen | AML, CML | SC |
t(8;14)(q24;q32) | 8 – MYC | ALL, NHL | DCDF |
14 – IGH@ | |||
t(11;14)(q13;q34) | 11 – CCND1 | NHL, PCM | DCDF |
14 – IGH@ | |||
t(14;18)(q32;q21) | 14 – IGH@ | NHL | DCDF |
18 – BCL2 | |||
t(*;14)(*.*;q32) | 14 – IGH@ | NHL, PCM | BAP |
del(13)(q14) or −13 | MIR16-1, MIR15A (CLL); unknown for PCM | CLL, PCM | SC, PP |
Trisomy 12 | 12 – 12cen | CLL | SC, PP |
unknown gene(s) | |||
del(11)(q23) | ATM | CLL | SC, PP |
del(17)(p13.1) | TP53 | CLL, PCM, NHL | SC, PP |
Acute Myeloid Leukemia
Approximately 40–60% of AML patients exhibit genetic aberrations that are readily detected by FISH, and in 2001, the World Health Organization (WHO) established an AML classification system that was based on recurrent genetic abnormalities; this was updated in 2008 [46] (see also Chap. 15 and Fig. 15.8). For each category, classical cytogenetics identifies the majority of aberrations; however, FISH may be used to detect cryptic abnormalities and variant rearrangements and to monitor disease states during and following treatment.
The t(8;21) juxtaposes the RUNX1 (AML1) gene on chromosome 21 and the RUNX1T1 (ETO) gene on chromosome 8. A dual color, dual fusion (DCDF) probe set has been developed to detect the fusion products on the derivative 8 and the derivative 21 chromosomes (Fig. 17.8). Similarly, a DCDF probe may be used for AML with t(15;17) in which there is a juxtaposition of the retinoic acid receptor alpha (RARA) gene at 17q21.1 and the PML (promyelocytic leukemia) gene at 15q24.1. 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 h) of the PML-RARA fusion is of utmost importance, so that patients may begin receiving 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, since patients with variant translocations may not respond to ATRA treatment. The t(11;17) and other RARA variants may be identified with a RARA break-apart (BAP) probe.
