Table of Contents- Lesson 1)
( Next ) (Glossary)
It is well known that DNA (deoxyribonucleic acid) is the blueprint of life. DNA provides the codes for the structural and enzymatic proteins that make up every cell. DNA is packaged into units called chromosomes. The chromosome number varies in different species. In humans there are 46 chromosomes, or 23 pairs of chromosomes (diploid), in every cell except the mature egg and sperm which have a set of 23 chromosomes (haploid). If the chromosomes in a single cell were stretched out and laid end to end, the DNA would be two meters long
Chromosomes are visible only during cell division, when the DNA is supercoiled and condensed to facilitate distribution into daughter cells. Cell division in somatic cells (mitosis) results in the creation of daughter cells with the same number of chromosomes as the original cell, a total of 46 chromosomes. Cell division in the germ cells, eggs and sperm (meiosis), results in the creation of daughter cells with half the number of chromosomes as the original cell, a total of 23 chromosomes. This reduction in the number of chromosomes is important so that the original number of 46 chromosomes is restored following fertilization of the egg by the sperm.
The chromosome constitution of an individual (karyotype) can be analyzed following tissue culture of an appropriate sample. The most commonly used sample is blood (using the white blood cells or lymphocytes) since it is the most accessible. However, other samples are used depending upon the indication: amniotic fluid cells, to analyze the karyotype of the fetus; products of conception, to analyze the cause of a miscarriage or stillbirth; bone marrow cells, to diagnose the presence or type of leukemia; and skin, to determine the presence of another cell line (mosaicism).
Since cells have to be grown in culture, it is important that samples are received in the laboratory within 24 to 48 hours after collection. The cells are grown in media for three days to two weeks depending on the sample source. Cell division is arrested during metaphase, when the chromosome material is condensed. Following hypotonic treatment and fixation, the cells are dropped on a slide and then stained. At least 20 metaphase spreads are analyzed and 2 or 3 metaphase spreads are photographed. The chromosomes are arranged to create a karyotype.
Chromosomes vary in size and in shape. The pairs
of autosomal chromosomes are arranged in a karyotype from the
biggest, #1, to the smallest, #22. The sex chromosomes are placed
to the right of the smallest autosomal chromosomes. Chromosomes
vary in shape depending upon the position of the centromere, the
structure that holds the two arms of the chromosomes together.
If the centromere is in the middle, the chromosome is metacentric
and the chromosome arms are equal in size. If the centromere is
off center, the chromosome is submetacentric with a short
arm labeled p (for petite) and a long arm labeled q (the next
letter after p). If the centromere is close to the end, the chromosome
is acrocentric and the very short arm consists of a stalk
and a knob (satellite). Based upon size and shape, chromosomes
are divided into eight groups: A (1 to 3), B (4 and 5), C (6 to
12), D (13 to 15), E (16 to 18), F (19 and 20), G (21 and 22)
and the sex chromosomes, XX in females and XY in males.
Fig. 1.1. Normal female karyotype
Chromosomes are further identified by banding patterns created by specialized staining procedures to produce G bands, R bands, C bands, etc. The choice of staining procedure depends upon the information that is desired. Most commonly, chromosomes are stained with trypsin-Giemsa to produce the G-banded pattern. The banding pattern is determined by the degree of chromatin condensation and the specific DNA-protein present at the different sites on the chromosome. These banding patterns are distinct and consistent for each chromosome. Thus, one can reliably identify the chromosome pairs (e.g., although of the same size and shape, the six acrocentric chromosomes in the D group can be differentiated into pairs 13, 14 or 15 based upon banding patterns). The bands are individually numbered (e.g., 11q23 refers to band #23 on the long arm q of chromosome #11).
The nomenclature of human chromosomes is based on several international consensus conferences. The convention is to first state the total chromosome number, followed by the sex chromosome constitution:
46,XX normal female
46,XY normal male
The description of abnormal karyotypes can be complicated. Examples of the more common designations are as follows:
del deletion
t translocation
dup duplication
ter terminal
ins insertion
mat maternal origin
i Isochromosome
pat paternal origin
inv inversion
+ additional chromosome
r ring chromosome
By convention, the total chromosome count and sex chromosomes are followed by a notation indicating the type of chromosome abnormality that is present. The abnormality is defined using one of the above designations followed by the number of the chromosome(s) that is involved, and the band(s) at the site of the breakpoint. See the examples below.
46,XY,del(22)(q21) a male with 46 chromosomes and a deletion on chromosome 22, with a breakpoint at band q21
46,XX,inv(7)(p11;q22) a female with 46 chromosomes and an inversion on chromosome 7, with breakpoints at bands p11 and q22
46,XX,t(1;6)(p23;q21) a female with 46 chromosomes and a translocation between chromosomes 1 and 6 with breakpoints at band p23 on the short arm of chromosome 1 and at band q21 on the long arm of chromosome 6
47,XX,+21 a female with an extra copy of chromosome 21, trisomy 21 or Down syndrome
Although G-banding remains the primary method of staining for chromosome analysis, even high resolution preparations may not clearly define the presence or absence of small deletions. In such cases, a relatively new staining method called fluorescence in situ hybridization (FISH) may provide useful diagnostic information. FISH makes it possible to visualize specific segments of DNA on metaphase chromosomes.
The specimen is processed and the slides are prepared in the usual manner. The chromosomes are denatured (i.e., DNA which is normally a double helix is rendered single-stranded) and a single-stranded DNA probe, that matches the segment of DNA on the chromosome that is thought to be missing, is added on the slide. Annealing takes place between the single-stranded probe and its complementary sequences on the single-stranded chromosomes. The excess probe is washed away and the slide is treated with other agents to permit visualization of the probe using fluorescence microscopy.
Given the specificity of hybridization of the probe to the complementary DNA sequence, the probe will only attach to a very specific segment of a particular chromosome. Thus, the probe GABR will only attach to the 15q11-12 band of the long arm of chromosome 15. This particular probe is used to define the deletion that is present in Prader-Willi syndrome (PWS).
In FISH, a control probe is used concurrently to label another segment of the chromosome under study. When testing for Prader-Willi syndrome, the control probe labels the tip of the long arm at band 15q21, "lighting up" both chromosome 15s at this site. Meanwhile, the GABR probe for PWS will light up the normal chromosome 15 and not the deleted chromosome 15 (positive test-deletion is present), or it will light up both normal chromosome 15s (negative test-no deletion). FISH is essential in the diagnosis of syndromes with microdeletions.
FISH can also be done using a mixture of several probes specific to different segments on the entire length of a chromosome. These are called painting probes. With these probes, one can "paint" the entire length of a specific chromosome. In cases of duplication, where extra chromosomal material is present, FISH is useful in identifying the origin of the extra material. Using chromosome paints on metaphase spreads, it is possible to label the chromosomes and chromosome segments from which the paint probes are derived (e.g., a FISH probe for chromosome 7 will label both chromosome 7s plus the duplicated chromosome 7 material on the same or on another chromosome). FISH is, therefore, useful in identifying the origin of duplications and marker chromosomes.
SUMMARY
There are 46 chromosomes, or 23 pairs of chromosomes, present in every cell except the eggs and sperm which have 23 chromosomes. Chromosomes 1 through 22 are referred to as autosomes. They are present in both males and females. The 23rd pair of chromosomes, referred to as the sex chromosomes, differ between the sexes. Females have two copies of the X chromosome, one from each parent. Males receive an X chromosome from the mother, and a Y chromosome from the father.
Chromosome analysis requires growing cells in culture and harvesting dividing cells. Chromosomes are arranged into a karyotype based on size, shape and banding pattern. Conventionally, twenty cells are analyzed for chromosome count and morphology.
Different staining techniques can be used to analyze
the chromosomes. Routine chromosome studies are done using trypsin-Giemsa
stain which results in the G-banding pattern. FISH studies are
done to identify microdeletions or the origin of extra chromosome
material.
1. How many chromosomes are present in a normal human cell?
2. List two reasons why FISH studies are done.
3. When ordering a chromosome study, why must the sample reach the laboratory within 24 to 48 hours?
Define the following notations:
4. 46,XX
1. 46
2. FISH studies are done when a syndrome caused by a microdeletion of chromosome material is suspected, or to identify the origin of extra chromosome material.
3. To do a chromosome study it is necessary to grow the cells in culture media for three days to two weeks depending on the sample source. Therefore, the sample must be received in the laboratory as soon as possible after it is collected so viable cells are available for study.