General Cytogenetics Information Basics

What Are Chromosomes?

Spanish Version

Cytogenetics is the study of chromosomes and the related disease states caused by abnormal chromosome number and/or structure. Chromosomes are complex structures located in the cell nucleus, they are composed of DNA, histone and nonhistone proteins, RNA , and polysaccharides. They are basically the "packages" that contain the DNA. Normally chromosomes can't be seen with a light microscope but during cell division they become condensed enough to be easily analyzed at 1000X. To collect cells with their chromosomes in this condensed state they are exposed to a mitotic inhibitor which blocks formation of the spindle and arrests cell division at the metaphase stage. A variety of tissue types can be used to obtain chromosome preparations. Some examples include peripheral blood, bone marrow, amniotic fluid, and products of conception. Although specific techniques differ according to the type of tissue used, the basic method for obtaining chromosome preparations is as follows: Sample log-in and initial setup. Tissue culture (feeding and maintaining cell cultures). Addition of a mitotic inhibitor to arrest cells at metaphase. Harvest cells. This step is very important in obtaining high quality preparations. It involves exposing the cells to a hypotonic solution followed by a series of fixative solutions. This causes the cells to expand so the chromosomes will spread out and can be individually examined. Stain chromosome preparations to detect possible numerical and structural changes.

Metacentric (Chromosome 1)

Chromosome Morphology
Under the microscope chromosomes appear as thin, thread-like structures. They all have a short arm and long arm separated by a primary constriction called the centromere. The short arm is designated as p and the long arm as q. The centromere is the location of spindle attachment and is an integral part of the chromosome. It is essential for the normal movement and segregation of chromosomes during cell division. Human metaphase chromosomes come in three basic shapes and can be categorized according to the length of the short and long

Submetacentric (Chromosome 9)

arms and also the centromere location. Metacentric chromosomes have short and long arms of roughly equal length with the centromere in the middle. Submetacentric chromosomes have short and long arms of unequal length with the centromere more towards one end. Acrocentric chromosomes have a centromere very near to one end and have very small short arms. They frequently have secondary constrictions on the short arms that connect very small pieces of DNA, called stalks and satellites, to the centromere. The stalks contain genes which code for ribosomal RNA. The diagrams to the left, called ideograms*, of G-banded chromosomes 1, 9, and 14 are typical examples of metacentric, submetacentric, and acrocentric chromosomes respectively. The ideogram is basically a "chromosome map" showing the relationship between the short and long arms, centromere (cen), and in the case of acrocentric chromosomes the stalks (st) and satellites (sa). The specific banding patterns are also illustrated. Each band is numbered to aid in describing rearrangements.

Acrocentric (Chromosome 14)

Chromosome Analysis
Virtually all routine clinical Cytogenetic analyses are done on chromosome preparations that have been treated and stained to produce a banding pattern specific to each chromosome. This allows for the detection of subtle changes in chromosome structure. The most common staining treatment is called Gbanding. A variety of other staining techniques are available to help identify specific abnormalities. Once stained metaphase chromosome preparations have been obtained they can be examined under the microscope. Typically 15-20 cells are scanned and counted with at least 5 cells being fully analyzed. During a full analysis each chromosome is critically compared band-for-band with it's homolog. It is necessary to examine this many cells in order to detect clinically significant mosaicism (see below). Following microscopic analysis, either photographic or computerized digital images of the best quality metaphase cells are made. Each chromosome can then be arranged in pairs according to size and banding pattern into a karyotype. The karyotype allows the Cytogeneticist to even more closely examine each chromosome for structural changes. A written description of the karyotype which defines the chromosome analysis is then made.

*Ideograms courtesy of Tim Knight, Fred Hutchinson Cancer Research Center, Seattle, WA.

Chromosome Abnormali ties

Normal Chromosomes
Normal human somatic cells have 46 chromosomes: 22 pairs, or homologs, of autosomes (chromosomes 1-22) and two sex chromosomes. This is called the diploid number. Females carry two X chromosomes (46,XX) while males have an X and a Y (46,XY). Germ cells (egg and sperm) have 23 chromosomes: one copy of each autosome plus a single sex chromosome. This is referred to as the haploid number. One chromosome from each autosomal pair plus one sex chromosome is inherited from each parent. Mothers can contribute only an X chromosome to their children while fathers can contribute either an X or a Y. Normal 550 and 650 Band Karyotypes

Chromosome Abnormalities
Although chromosome abnormalities can be very complex there are two basic types: numerical and structural. Both types can occur simultaneously. Numerical abnormalities involve the loss and/or gain of a whole chromosome or chromosomes and can include both autosomes and sex chromosomes. Generally chromosome loss has a greater effect on an individual than does chromosome gain although these can also have severe consequences. Cells which have lost a chromosome are monosomy for that chromosome while those with an extra chromosome show trisomy for the chromosome involved. Nearly all autosomal monosomies die shortly after conception and only a few trisomy conditions survive to full term. The most common autosomal numerical abnormality is Down Syndrome or trisomy-21. Trisomies for chromosomes 13 and 18 may also survive to birth but are more severely affected than individuals with Down Syndrome. Curiously, a condition called triploidy in which there is an extra copy of every chromosome (69 total), can occasionally survive to birth but usually die in the newborn period. Another general rule is that loss or gain of an autosome has more severe consequences than loss or gain of a sex chromosome. The most common sex chromosome abnormality is monosomy of the X chromosome (45,X) or Turner Syndrome. Another fairly common example is Klinefelter Syndrome (47,XXY). Although there is substantial variation within each syndrome, affected individuals often lead fairly normal lives. Occasionally an individual carries an extra chromosome

which can't be identified by it's banding pattern, these are called marker chromosomes. The introduction of FISH techniques has been a valuable tool in the identification of marker chromosomes. Structural abnormalities involve changes in the structure of one or more chromosomes. They can be incredibly complex but for the purposes of this discussion we will focus on the three of the more common types: Deletions involve loss of material from a single chromosome. The effects are typically severe since there is a loss of genetic material. Inversions occur when there are two breaks within a single chromosome and the broken segment flips 180 (inverts) and reattaches to form a chromosome that is structurally out-of-sequence. There is usually no risk for problems to an individual if the inversion is of familial origin (has been inherited from a parent.) There is a slightly increased risk if it is a de novo (new) mutation due possibly to an interruption of a key gene sequence. Although an inversion carrier may be completely normal, they are at a slightly increased risk for producing a chromosomally unbalanced embryo. This is because an inverted chromosome has difficulty pairing with it's normal homolog during meiosis, which can result in gametes containing unbalanced derivative chromosomes if an unequal cross-over event occurs. Translocations involve exchange of material between two or more chromosomes. If a translocation is reciprocal (balanced) the risk for problems to an individual is similar to that with inversions: usually none if familial and slightly increased if de novo. Problems arise with translocations when gametes from a balanced parent are formed which do not contain both translocation products. When such a gamete combines with a normal gamete from the other parent the result is an unbalanced embryo which is partially monosomic for one chromosome and partially trisomic for the other.

Numerical and structural abnormalities can be further divided into two main categories: constitutional, those you are born with; and acquired, those that arise as secondary changes to other diseases such as cancer. Sometimes individuals are found who have both normal and abnormal cell lines. These people are called mosaics and

in the vast majority of these cases the abnormal cell line has a numerical chromosome abnormality. Structural mosaics are extremely rare. The degree to which an individual is clinically affected usually depends on the percentage of abnormal cells. A routine Cytogenetic analysis typically includes the examination of at least 15-20 cells in order to rule out any clinically significant mosaicism. These are just some of the more common abnormalities encountered by a Cytogenetic Laboratory. Because the number of abnormal possibilities is almost infinite, a Cytogeneticist must be trained to detect and interpret virtually any chromosome abnormality that can occur.

Examples of Chromosome Abnormalities

Click on any of the following links to see some examples of numerical and structural chromosome abnormalities: Example 1: Down Syndrome, a common numerical abnormality. Example 2: An inversion in chromosome 10. Example 3: An interstitial deletion of chromosome 16. Example 4: A translocation between chromosomes 2 and 15. Example 5: A translocation between chromosomes 5 and 8. Example 6: A subtle inversion in chromosome 3. Example 7: An interstitial deletion of chromosome 7. Example 8: An unbalanced translocation between chromosomes 13 and 14.

Fluorescent IN SITU Hybridization (FISH)

Version

Spanish

Fluorescent IN SITU Hybridization (FISH) is a relatively new technology utilizing fluorescently labeled DNA probes to detect or confirm gene or chromosome abnormalities that are generally beyond the resolution of routine Cytogenetics. The sample DNA (metaphase chromosomes or interphase nuclei) is first denatured, a process that separates the complimentary strands within the DNA double helix structure. The fluorescently labeled probe of interest is then added to the denatured sample mixture and hybridizes with the sample DNA at the target site as it reanneals (or reforms itself) back into a double helix. The probe signal can then be seen through a fluorescent microscope and the sample DNA scored for the presence or absence of the signal.

Metaphase FISH
FISH can be used in metaphase cells to detect specific microdeletions beyond the resolution of routine Cytogenetics or identify extra material of unknown origin. It can also help in cases where it is difficult to determine from routine Cytogenetics if a chromosome has a simple deletion or is involved in a subtle or complex rearrangement. In addition, metaphase FISH can detect some of the specific chromosome rearrangements seen in certain cancers. The number of microdeletion syndromes diagnosed by FISH is expanding rapidly. The sensitivity of these tests in every case is better than routine Cytogenetics but depends on the particular syndrome. In some syndromes, the probe is specific for the defective gene as in Williams Syndrome where a deletion has been shown in the elastin gene in 96% of individuals with a firm diagnosis. In other syndromes such as Prader-Willi/Angelman, the etiology of the syndrome is heterogeneous and microdeletions compose only a portion of the cases (60%).

Microdeletion Syndromes Currently Diagnosable with FISH

Cri-du-Chat Miller-Dieker Syndrome Smith-Magenis Syndrome Steroid Sulfatase Deficiency DiGeorge/Velo-CardioFacial/CATCH-22/Shprintzen Syndrome Kallman Syndrome Williams Syndrome Wolf-Hirschhorn Prader-Willi/Angelman Syndrome

Interphase FISH
FISH can be used in interphase cells to determine the chromosome number of one or more chromosomes as well as to detect some specific chromosome rearrangements that are characteristic for certain cancers. The primary advantage of interphase FISH is that it can be performed very rapidly if necessary, usually within 24 hours, because cell growth is not required. A good example is the Aneuploid Screen test which is performed on amniotic fluid cells when there is a strong clinical indication for one of the common trisomies. The sample nuclei are denatured and hybridized with DNA probes for chromosomes 13, 18, 21, X, and Y and results usually obtained within 24 hours. Routine Cytogenetics is included with an Aneuploid Screen to confirm the results or detect any abnormalities not detected by interphase FISH.

Click on the following links to see some FISH examples:

Interphase FISH Examples

This is an example of the Aneuploid Screen test where interphase nuclei from amniotic fluid cells have been combined with DNA probes for chromosomes 13, 18, 21, X, and Y. The nucleus on the left has been hybridized to probes for chromosomes 13 (green), and 21 (red). The nucleus on the right has been hybridized to probes for chromosomes 18 (aqua), X (green), and Y (red). Since there are two signals each for chromosomes 13, 18, and 21 and one signal each for the X and Y chromosome this fetus is a normal male with respect to the Aneuploid Screen test.

This Aneuploid Screen test shows a female fetus with trisomy-21. The nucleus on the left has been hybridized to probes for chromosomes 13 (green), and 21 (red) and clearly has three red signals. The nucleus on the right has been hybridized to probes for chromosomes 18 (aqua), X (green), and Y (red). Since it has two green signals and no red signal it is female.

Metaphase FISH Examples

This is an example of a metaphase cell that has been hybridized with the probe for DiGeorge/Velo-Cardio-Facial/CATCH 22/Shprintzen Syndrome which is caused by a microdeletion on chromosome 22. The probe in this particular case is a dual-color mixture of two seperate probes for chromosome 22. The green signal is an internal control and is located at 22q13. It allows for quick identification of both #22 chromosomes. The red signal is located at the DiGeorge region at 22q11.2. Since both 22's have the red signal in this cell there is not a microdeletion within the DiGeorge region and this individual would not have DiGeorge Syndrome.

This metaphase has been hybridized with a "painting" probe for chromosome 4 which causes the entire chromosome to fluoresce. One chromosome 4 from this individual was abnormal but it was difficult to determine from routine Cytogenetics if it had a small terminal deletion at 4q or was the result of a more complex rearrangement. Since both 4's are fluorescent along their entire length and no fluorescent material is present on any other chromosome, this suggests that the abnormality is a small terminal deletion. The metaphase below is from the same individual and further confirms this diagnosis.

This metaphase from the same individual as the cell above has been hybridized with a probe for the terminal part of chromosome 4q. Since there is only one green signal this confirms that one chromosome 4 is missing material from the terminal end of 4q. This case is a good example of how routine Cytogenetics and FISH can be used together to accurately diagnose subtle chromosome abnormalities.

This is an example of a metaphase cell that has been hybridized with the probe for Steroid Sulfatase Deficiency which is caused by a microdeletion on the X chromosome. The probe in this particular case is a mixture of two seperate probes for the X chromosome, both red in color. The "X cen" probe signal is an internal control and is located at the X centromere. It allows for quick identification of the X chromosome(s). The "Xp22.3" probe signal is located at the Steroid Sulfatase region at Xp22.3. Since there are two X chromosomes and only one has the Steroid Sulfatase gene signal, this individual is a female carrier for Steroid Sulfatase Deficiency.