|The past 20 years has seen an explosion
of interest in the study of human chromosomes (cytogenetics) primarily
as a result of two events. First was the development of a simple technique
for producing good preparations of chromosomes from readily available
tissue (e.g. white blood cells). This technique was made possible
by the discovery that these cells could be stimulated to divide in
culture and blocked at metaphase (the stage when the chromosomes can
be most readily identified). Applications of this technique quickly
led to the conclusion that the normal human chromosome number is 46.
That is, the somatic cells (e.g. skin cells, white blood cells) have
46 chromosomes while the gametes (e.g. sperm, ova) have 23. Table
1 shows that humans have an' intermediate number of chromosomes relative
to other organisms.
TABLE 1. Diploid chromosome numbers of various organisms.
# of Chromosomes
|Bread Mold (Neurospora)
|Toad (Bufo americans)
|Frog (Rana pipiens)
The second major reason for interest in human chromosomes was a
report in 1959 that cells from people with Down's syndrome (mongolism)
contained an extra chromosome. This was the first known association
between a chromosomal abnormality and human disease. It set off
a search which has resulted in the discovery of a wide variety of
chromosomal abnormalities ranging from interchanged chromosome sections
with no clinical effects to minute changes that are lethal.
Most numerical chromosomal abnormalities arise from failure of
a pair of homologous chromosomes to separate properly during meiosis
(nondisjunction). As a result, one of the daughter cells has one
or more extra chromosomes and the other is lacking an equivalent
number. In humans a loss of chromosomal material is more lethal
than a gain. Turner's syndrome (females with only one X chromosome)
provides the only known example of humans surviving with less than
the normal number of chromosomes.
There are some other disorders such as "cri du chat"
syndrome (named from the observation that newborn infants with this
disorder have an unusual cry which sounds like a kitten's meow)
where a person can survive with part of a chromosome missing. Loss
or gain of chromosomal material is frequently but not always associated
with mental retardation. Chromosomal abnormalities probably account
for about 10 to 15 percent of all cases of mental retardation.
It is important to note some of the basic differences between single
gene disorders such as phenlyketonuria (an inability to metabolize
a particular amino acid (phenylalanine)) which leads to severe mental
retardation if untreated and chromosomal disorders such as Down's
syndrome. One of the major functions of genes is to supply the proper
amino acid sequence for assembling proteins. A useful analogy is
to think of the chromosomes as serving as a dictionary for the cell.
When we want to assemble the letters of the alphabet into a particular
word, we look up the correct sequence in the dictionary. Similarly,
each structural gene tells the cell the proper sequence in which
to assemble the amino acids in order to make a particular protein
molecule. Each normal human somatic cell contains 23 pairs of chromosomes,
one member of each pair is from the father and the other from the
mother. In essence, the diploid cell has two similar dictionaries,
one from each parent. A disorder due to a single recessive gene
is like having a misprint in one word of the dictionary. For such
a disorder to appear, both parents must have at least one form of
the gene (allele) which carries the wrong instructions. The Punnett
square (Fig. 1) shows that if both parents are carriers of the disease
(i.e., one allele carries the correct instructions, P, for a certain
protein and the other allele carries the wrong instructions, p)
there is one chance in four that the zygote will receive no correct
instructions for the protein and will therefore develop the disease.
Figure 1. Punnett square showing the pattern of
inheritance from parents carrying a recessive gene.
In some cases the amount of a particular protein produced
appears to be proportional to the number of correct instructions.
Thus, the normal person (PP) with two sets of correct instructions
would be expected to have twice as much of this particular protein
as the carrier (Pp) with only one set of correct instructions. Generally,
the decrease found in the carrier does not result in any clinical
symptoms although it can often be detected with special chemical
tests. In other words, PP and Pp may appear- to be the same physically,
but we can often distinguish between them chemically.
The chromosomal disorders differ from single gene disorders in
that the parents rarely are carriers of any defect and the basic
problem is too few or too many normal genes rather than an abnormal
gene. Having an extra chromosome is like having an extra page in
our dictionary. The additional instructions (genes) supplied by
the extra chromosome would be expected to cause abnormally high
production of some proteins. This may have a severe effect on the
development of the zygote as can be seen by the fact-that the presence
of an extra chromosome 21 (trisomy) causes the symptoms associated
with Down's syndrome. Trisomy of the larger chromosomes, which presumably
carry even more genes, is usually not compatible with life. The
sex chromosomes are an exception as it is possible for a person
to have extra sex chromosomes with relatively little known effect.
Many chromosomal abnormalities arise from errors occurring during
cell division. The fact that Down's syndrome occurs about once in
every 700 births suggests that these errors are not extremely rare.
A number of such errors probably occur throughout our bodies each
day. However, it is only those which occur during formation of gametes
that are regularly observed since they can be passed on to the offspring.
Translocations represent a special type of chromosomal
abnormality which may be transmitted through a family. In this situation,
parts of two different chromosomes become joined. A person can carry
a translocation and be otherwise normal but it may be difficult,
or sometimes impossible, to produce gametes with the correct amount
of genetic information.
Human metaphase chromosomes have characteristic shapes as shown
in the sample karyotype. In Figure 5 they appear to be double except
at the region of constriction, the centromere. The various "V"
and "X" shapes depend on the length of the chromosome
and the position of the centromere. A "V" shape results
when the centromere is near an end, and such chromosomes are called
"acrocentrics". The "X" shaped chromosomes,
with their centromeres in the middle, are called "metacentric".
As shown in Figure 2 the short arm of a chromosome is referred to
as "p" and the long arm as "q". Chromosomes
can be classified according to their "centromeric index"
which can be thought of as the percentage of the total length contributed
by the short arm.
In "banded" preparations such as shown in Figure
6, chromosomes have a characteristic pattern of dark and light
bands. Darkly staining regions are called "positive bands".
"Negative bands" do not stain and appear as light
regions. Some regions stain in chromosomes of some individuals
but not in others and are called "variable bands"
Figure 2. Banding pattern of human chromosome #1.
Preparation of a standard diagnostic karyotype involves cutting
the individual chromosomes out of an enlarged photograph of a dividing
cell (Figure 4, 6). Each chromosome is then matched with the other
member of its pair and fixed onto its proper position on a standard
karyotype form. The karyotype form organizes the chromosomes into
7 groups (A through G) based on size, shape, centromeric index,
and banding pattern if available (Figure 5, 7). Any numerical or
major structural abnormalities will be obvious at this stage.
Although karyotype analysis is useful for clinical diagnosis of
certain diseases it is most often used in conjunction with amniocentesis
(Figure 3) to determine the chromosomal make-up of a fetus before
birth. It is now possible to diagnose many genetic diseases prenatally
and this raises important ethical and moral questions regarding
the termination of pregnancy involving an abnormal fetus.
Figure 3. Amniocentesis. A sample of amniotic fluid (mostly fetal
urine and other sections) is taken by inserting a needle into the
amniotic cavity during or about the 16th week of gestation. The
fetal cells are separated from the fluid by centrifugation. The
cells are then cultured so that a number of biochemical, enzymatic,
and chromosomal analyses can be made.
Figure 4. Metaphase spread of unbanded chromosomes
from a normal male (XY).
Photo Credit: Carolina Biological Supply Company
Figure 5. Unbanded karyotype of a normal male subject.
(Prepared from Figure 4)
Figure 6. Metaphase spread of banded chromosomes
from a normal female (XX).
Photo Credit: Carolina Biological Supply Company.
Figure 7. Banded karyotype of a normal female subject:
(Prepared from Figure 6)
Preparation of Chromosomes for Karyotype Analysis
Chromosomal preparations are most easily obtained from white blood
cells (lymphocytes). The preparation is set up by placing a few
drops of blood in a tube containing growth medium supplemented with
phytohemagglutinin to stimulate the lymphocytes to divide. The tube
is incubated at 37°C. After 72 hours colchicine is added and
incubation is continued for another 1 1/2 hours. Colchicine will
arrest dividing cells at metaphase.
At metaphase, each chromosome has replicated itself with the two
strands joined at the centromere. If cell division had continued,
the strands would have pulled apart and one strand would have entered
each of the new daughter cells. Thus, the X-shaped structures seen
in the photographs would have become two chromosomes if they had
been allowed to separate.
The chromosomal preparation is centrifuged to separate the cells
and then treated with a hypotonic solution to destroy the red cells
and make the lymphocytes easier to rupture. The lymphocytes are
then fixed in a mixture of methanol and glacial acetic acid. The
fixed cells are dropped on a cold slide and air dried to rupture
the cells and spread the chromosomes. The slide is washed and stained
with Giemsa. Treatment of the chromosomes with enzymes or mild acid
before staining causes a characteristic banding pattern for each
chromosome. Such "banded" preparations allow more precise
matching of chromosome pairs and identification of structural abnormalities.
Compare the banded and unbanded karyotypes of Figure 5 and 7.
While the chromosomes are visible under low power (100X), counting
and photography are routinely done under oil immersion (1000X).
The chromosomes in at least 25 cells are usually counted to be reasonably
certain of detecting any chromosomal abnormalities. One or two well-spread
fields are then photographed for karyotyping.
Genetic Conditions Detectable by Karyotype Analysis
Cri-du-chat Syndrome (5p deletion) A chromosomal abnormality
caused by deletion of all or part of the short arm of chromosome
5. The most distinctive characteristic of this syndrome is the infant's
cry, which is identical to that of a mewing kitten. In addition,
there are characteristic abnormalities which include a small head,
moon-like face, and receding lower jaw. Affected individuals are
severely retarded. Fatality is low, and many individuals survive
well into adulthood. In most cases, the deletion appears for the
first time in the affected individual (the parents being normal).
Down Syndrome (Trisomy 21) A condition caused by an extra
chromosome 21. Individuals with Down Syndrome are usually of short
stature, with a broad skull, round face, epicanthic fold, and mental
retardation. About 1 in 6 children with this condition die within
the first year, usually of congenital heart defects. The frequency
of leukemia is 10-14 times higher in these individuals than in the
population at large. The average life expectancy is about 30 years.
Down Syndrome occurs with a frequency of 1 in 700 births, and is
directly related to maternal age. 40% of Down Syndrome patients
are born to women over 40, although they produce only about 4%0
of all births.
Edwards Syndrome (Trisomy 18) A condition associated with
an extra for chromosome 18. Characterized by congenital malformations
which affect many organ systems. Low set, faunlike ears with pointed
pinnae are characteristic. The hands are usually tightly closed
in fists, with the index finger folded to overlap the third digit,
and the fifth overlapping the fourth digit. Cardiac malformations
include septal defects. Average survival is 2-3 months for males
and 10 months for females.
Patau Syndrome (Trisomy 13) A condition caused by an extra
for chromosome 13. The phenotype is somewhat variable, but includes
mental retardation, cleft lip and/or palate, deafness, cardiac malformations,
and posterior protrusions of the heel. Most children with this condition
die within the first three months of life. This condition, caused
by chromosome nondisjunction, occurs with a frequency of 1 in n
Turner Syndrome (45,X) The first chromosomal anomaly involving
the sex chromosomes to be described. Affected individuals are females,
having only one X chromosome. The phenotypic characters include
small stature, swelling of the hands and feet as infants, and an
excess of skin at the nape of the neck. Overall, the symptoms are
not usually severe, and the survival rate is not different from
the normal population. This condition, arising from chromosomal
nondisjunction, occurs with a frequency of 1 in 3000 female births.
Wilms Tumor A chromosomal abnormality associated with a
deletion within the short arm of chromosome 11. This internal deletion
results in aniridia (absence of the iris), mental retardation, microcephaly,
and malignant tumors of the kidney. Tumor growth usually occurs
in infants and young children, and leads to death.
Klinefelter Syndrome (47,XXY) A chromosomal abnormality
caused by the presence of two X chromosomes in addition to one Y
chromosome. Individuals with this condition are phenotypically male,
but often show tendency toward female secondary sexual characteristics,
such as enlarged breasts. Diagnosis is usually at puberty. Individuals
are sterile. Intellectual development is in the normal range, and
survival is also within the normal range. Chromosomal nondisjunction
is the cause of this condition, which occurs with a frequency of
2 in 1000 male births. In a small percentage of cases, more complex
karyotypes such as 48, XXXY are seen. In general, higher numbers
of-sex chromosomes are associated with the development of mental
XXX Syndrome. The 47,XXX female karyotpye was first described
in 1959 and has been shown to occur in about 1 in 1200 births. In
most cases, the phenotype is completely normal, including fertility.
There is a slight disturbance in intellectual development in two-thirds
of the cases, but otherwise, the addition of an extra chromosome
has little or no effect on the individual. Normality is attributed
to the inactivation of two X chromosomes (as Barr bodies) in somatic
XYY Syndrome. This chromosomal condition was first described
in 1961, and subsequent population surveys revealed that 7 XYY males
were detected in a group of 197 mentally retarded males institutionalized
for violent or criminal tendencies. On this basis, it was suggested
that the presence of an extra Y chromosome might predispose individuals
toward aggressive behavior. XYY is a common condition, occurring
with a frequency of 1 in 1000 male births, and only a small percentage
of these individuals (about 3.5%) are ever institutionalized. A
wide range of phenotypes is present in this condition, but only
two, tallness and subnormal intelligence, are constant.
Carefully cut out the individual chromosomes from the photographed
metaphase spread. Using the sample karyotype in Figure 7 as a guide,
arrange each chromosome on the blank karyotype form. Do not fasten
the chromosomes to the karyotype form or throw away any paper scraps
until you have identified each chromosome.
The A group consists of six chromosomes. The number 1 pair is the
longest and has the centromere in the middle. Number 2 is the second
longest and has the centromere slightly off centre Number 3 is the
third longest with the centromere in the middle. The B group consists
of four long chromosomes with the centromeres very close to one
end. If the preparation is not banded, there is no way to determine
which two belong to pair 4 and which is pair 5. The C group consists
of 14 medium-sized chromosomes with the centromeres slightly off-center.
The X chromosome also falls in this group. Therefore, a male will
have 15 C-type chromosomes and a female will have 16. Frequently,
the X chromosome is the largest in the C group. The D group consist
of six chromosomes slightly smaller than the C's with the centromeres
very near one end. The E group resembles the C group but the chromosomes
are much smaller. The F group chromosomes are very small with the
centromeres in the middle. The G group includes the four smallest
chromosomes with-the centromeres so close to the ends that it is
difficult to see any short arms at all. The Y chromosome falls into
the G group. Therefore, a male will have five G-type chromosomes
and a female four. The Y chromosome can often be separated from
the other members of the G-group by its parallel long arms if unbanded.
When you have completed the normal karyotype attempt one of the
abnormal chromosome complements. First, count the chromosomes. The
normal number is 46. If there are more than 46 chromosomes you know
there is extra chromosomal material. If there are less than 46,
there are two possibilities: either a chromosome has been lost or
two chromosomes have joined. Now, cut out the chromosomes and prepare
the karyotype by matching the chromosome pairs. Remember, a female
has two X chromosomes and a male has one X and one Y chromosome.
Compare your abnormal karyotype with a normal one to identify the
type of abnormality.