Molecular Cytogenetics of Human Diseases

One of the most effective tools for teaching biological concepts is to provide human examples to which all students can readily relate. In the field of genetics, this comes naturally due to the many human traits that have a genetic etiology. Many human genetic disorders have a chromosomal basis which can be demonstrated to students by using a technique called fluorescence in situ hybridization, or FISH. These chromosomal abnormalities include aneuploidies (trisomies, monosomies), deletions, duplications, translocations, and inversions. A vast selection of normal and chromosomally aberrant cell cultures are available through several different cell line repositories. Two of the major facilities are the National Institute of General Medical Sciences (NIGMS) Human Genetic Mutant Cell Repository, and the American Type Culture Collection (ATCC). The cell lines may be obtained as either fibroblast or lymphoblast cultures.

There are a number of different laboratory exercises that can be designed to illustrate the concept of the chromosomal basis of human genetic diseases. A few examples are provided below, all of which rely on the use of the FISH technology. Each exercise opens up the opportunity to expand the classroom discussion to include broader topics such as the Human Genome Project, prenatal diagnosis and reproductive technology, ethical and legal issues, and eugenic concerns.

In situ hybridization is a technique that allows highly sensitive detection of specific nucleic acid sequences (DNA or RNA) in specimens fixed on a microscope slide. The specimen may be chromosome spreads, cells, or tissue sections. Until recently, in situ hybridization was performed using radioactive labeled probes. The transition to using fluorescent labels has vastly broadened the applications and increased the usage of the technology. Use of fluorescent labels for in situ hybridization is referred to as FISH. The major advantages to fluorescent probes include: safety, increased spatial resolution, reduced turn around time for results, and the capability of simultaneous detection of multiple DNA regions of interest by using different combinations of fluorochrome labeled probes.

There are many different applications of FISH in mammalian cytogenetics. The major categories of applications include:

There are two main types of probe labeling systems for FISH: direct and indirect. In the direct method, the fluorochrome molecule is bound directly to the nucleotides of the probe. This allows visualization of the probe signal immediately following the hybridization reaction and wash steps. In the indirect labeling method, hapten molecules are attached to the nucleotides which are detected by a secondary molecule conjugated to a fluorochrome. Two of the most popular detection systems are the digoxygenin-antidigoxygenin system and the biotin-strepavidin system.

Digoxygenin (dig) is a steroid isolated from digitalis plants (Digitalis purpurea and Digitalis Lanata). Since the blossoms and the leaves of these plants are the only naturally occurring sources of this compound, the anti-dig antibody is highly specific for its target. Background hybridization is minimal. The anti-dig antibody can be conjugated with a variety of different fluorochromes, such as fluorescein (FITC), Texas Red, and rhodamine. These molecules can emit a distinct visible color upon excitation by a fluorescence or confocal microscope.

Alternatively, biotin may be conjugated to the base of nucleotides. Strepavidin has a very high binding capacity for biotin. Strepavidin may be conjugated to a variety of different fluorochromes.

The probes can be prepared in various ways. Two of the most common methods are referred to as nick translation and polymerase chain reaction (PCR). For nick translation, several companies sell nick translation kits that contain all the necessary reagents. Briefly, the procedure involves treating the DNA source with Dnase I to generate single stranded nicks along the double stranded DNA molecule. The 5’ to 3’ exonuclease activity of DNA polymerase I enzyme removes additional nucleotides to generate gaps. The same enzyme replaces the excised nucleotides with "labeled" nucleotides using the other complementary intact strand as template. The "labeled" nucleotides may contain a digoxigenin or biotin conjugated base for the indirect labeling method, or a fluorochrome conjugate such as fluorescein, rhodamine, or Texas Red for direct labeling.

The second method of preparing the probe uses PCR. Specific primers must be available to amplify the DNA sequences of interest. The primer sequences can be synthesized to order through a variety of different vendors. The nucleotides added to the PCR reaction may contain a digoxigenin or biotin conjugated base for the indirect labeling method, or a fluorochrome conjugate for direct labeling.

Hybridization involves the reannealing (also called renaturation) of denatured single stranded DNA to their complementary sequences. In FISH, the probe must anneal with its complementary sequence on the chromosome before the original complementary strand does. There are a number of factors that effect this process. They include:

There are a wide variety of human disorders that are due to an abnormal number of human chromosomes. Trisomies and monosomies are the major types of anueploidy that are seen in live births. Down syndrome (trisomy 21), Klinefelter syndrome (XXY), and Turner’s syndrome (monosomy X) serve as classic examples of chromosomal disorders. Karyotypes generated from cells of individuals with these conditions using FISH technology and analyzed on a fluorescence or confocal microscope enhance the visualization of the aneuploidy concept. Instructors can have students compare their own normal karyotypes generated from white blood cell cultures with that of karyotypes generated from individuals with various chromosomal disorders. All of these genetic conditions provide an ideal avenue of discussion leading to the Human Genome Project, prenatal diagnosis technology, ethical issues, and eugenic concerns.

A. Down Syndrome

Down syndrome is one of the most common genetic causes of mental retardation. The clinical manifestations of the condition include congenital heart defect, shortened stature, mental retardation, protruding tongue, epicanthic fold of eyelids, hypotonia, and increased risk of leukemia and Alzheimer’s disease with increased age. In most cases, the cytogenetic basis of the disease is the presence of three chromosome #21 (trisomy 21). In a much smaller percentage of cases (about 5%), the condition may be caused by a translocation involving chromosome 21 and another chromosome.

To demonstrate that Down syndrome is associated with three copies of chromosome 21, students may prepare chromosome spreads from lymphocyte or fibroblast cell cultures obtained from Down syndrome patients available from several different cell culture repositories. Whole chromosome #21 probes for FISH analysis are available from ONCOR, Inc. (Gaithersburg, MD) or Vysis, Inc. (Downers Grove, IL). The recognition of each chromosome 21 in the cells, even in interphase nuclei, is made very easy by the distinctive coloration of the probe. This eliminates the need for the students to learn to recognize banding patterns to differentiate each chromosome.

The following protocol for detection of trisomy 21 using FISH is based on a whole chromosome paint (WCP) #21 Spectrum Green kit available from VYSIS, Inc. For normal control, students may prepare their own white blood cell cultures, as described below, or obtain a normal cell line from a cell repository center.

(Note: The formula for preparing all the reagents that are underlined in the procedure can be found in the "Solutions" section.)

1. White Blood Cell Cultures (for students who want to use their own cells as normal control)

3. Slide preparation for FISH

4. Probe preparation

7 ul hybridization buffer

1 ul WCP DNA probe for chromosome #21

2 ul water

5. Hybridization

6. Wash and Detection

#1, #2, #3 = 50% formamide/2XSSC

#4, #5, #6 = 2XSSC

Klinefelter syndrome is due to the presence of an extra X chromosome in the male, resulting in a karyotype of 47, XXY. The frequency of the condition is approximately 1 in 1000 male births. Clinical manifestations of the syndrome do not develop until puberty, at which time they include poor sexual development with very low fertility, some degree of breast development, and subnormal intelligence in some affected individuals.

Lymphocyte and fibroblast cell cultures from individuals with Klinefelter syndrome are available from several different cell culture repositories. Detection of the aberrant karyotype can be conducted by setting up a dual label FISH experiment using the X and Y chromosome alpha satellite probes available through ONCOR, Inc. For the normal control, students may prepare their own white blood cell cultures, or obtain a normal cell line from a cell repository center.

The following protocol for detection of Klinefelter syndrome using FISH is based on DNA probes for the X alpha satellite (biotin-labeled) and Y centromere region (dig-labeled) available from ONCOR, Inc. Comparable probes are available through VYSIS, Inc., with slight modifications as described in their kit.

1. White Blood Cell Cultures (for students who want to use their own cells as normal control) see under section "A. DOWN SYNDROME".

2. Preparation of Chromosome Spreads on Slides

see under section "A. DOWN SYNDROME".

3. Slide preparation for FISH

4. Probe Preparation

8 ul hybridization mix

1 ul herring sperm DNA

1 ul probe (1:1 premix of X alpha satellite probe and Y centromere (cocktail) probe)

DO NOT PREANNEAL AFTER THE DENATURATION

5. Hybridization

6. Wash and Detection

#1, #2, #3 = 50% formamide/2XSSC

#4, #5, #6 = 2XSSC

Chromosomal deletions can occur in many different sizes anywhere within the genome. Certain deletions in specific regions of chromosomes lead to specific syndromes that have been well characterized. One example is the Prader-Willi syndrome associated with a deletion in the chromosome 15q11-13 region. Clinically, affected individuals develop an uncontrollable compulsion to eat starting at about five years of age. This leads to obesity and related health problems such as diabetes. Other symptoms include mental retardation, poor sexual development, and behavioral problems. The occurrence of this condition is estimated to be approximately 1 in 10,000 to 1 in 25,000 people. There is a greater preponderance of males affected with this condition.

Lymphocyte and fibroblast cell cultures from individuals with Prader-Willi syndrome are available from several different cell culture repositories. Detection of the aberrant karyotype can be conducted by using a specific fluorescent probe available through several different companies. Students can use their own white blood cell cultures as a normal control, or purchase a normal cell line from a cell repository.

The following protocol for detection of Prader-Willi syndrome using FISH is based on the D15S11 (dig-labeled) DNA probe available from ONCOR, Inc. Comparable probes are available through VYSIS, Inc., with slight modifications as described in their kit.

1. White Blood Cell Cultures (for students who want to use their own cells as normal control) see under section "A. DOWN SYNDROME".

2. Preparation of Chromosome Spreads on Slides

see under section "A. DOWN SYNDROME".

3. Slide preparation

4. Probe preparation

DO NOT PREANNEAL.

5. Hybridization

6. Wash and Detection

#1, #2, #3 = 50% formamide/2XSSC

#4, #5, #6 = 2XSSC

Note: from this point on, avoid exposure of the slide to direct light. Use dim lighting conditions or lamps facing away from the slide.

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