Since chromosomes are visible as individual
structures under the light microscope only
during metaphase (or under special conditions
and for certain purposes, during prometaphase),
every chromosome analysis requires
dividing cells. In vivo, only bone marrow cells
contain a sufficient proportion of cells in mitosis.
Thus, in-vivo chromosome analysis of cells
is limited to bone marrow. All other procedures
for analyzing chromosomes in mitosis require
culturing of suitable cells (cell culture). Most
commonly, lymphocytes from blood are cultured
for chromosome preparations.
Peripheral blood lymphocytes stimulated by
phytohemagglutinin grow in a suspension culture.
Their life span is limited to a few cell divisions.
However, by exposing the culture to Epstein-
Barr virus they can be transformed into a
lymphoblastoid cell line with permanent
growth potential. Such cultures arewidely used
because they are much easier to handle than adhesion
cultures (p. 122).
In addition, fibroblasts from a piece of skin can
be propagated in cell culture for analysis (see
p. 122). However, since this procedure is somewhat
elaborate and time-consuming, it is used
only for certain purposes.
Sunday, April 12, 2009
Chromosome analysis from blood
For the cell culture, either peripheral blood is
used directly or lymphocytes are isolated from
peripheral blood (T lymphocytes). A sample of
about 2ml of peripheral blood is needed. The
blood is prevented from clotting by use of a heparinized
syringe, since clumping of the blood
cells precludes culturing (the proportion of heparin
to blood is about 1: 20). Peripheral blood
or isolated lymphocytes are placed in a vessel
with culture medium. The cells are generally
stimulated with phytohemagglutinin, a protein
from plants that unspecifically stimulates
lymphocytes to divide. The culture requires
about 72 hours at 37!C for cells to divide.
Lymphocyte cultures are suspension cultures;
i. e., the cells divide in culture medium without
attaching to the culture vessel. Cell division is
arrested and the culture is terminated by
adding a suitable concentration of a colchicine
derivative (colcemid) two hours prior to
harvest. Colcemid interrupts mitosis during
metaphase, so that a relative enrichment of
cells in metaphase results.
used directly or lymphocytes are isolated from
peripheral blood (T lymphocytes). A sample of
about 2ml of peripheral blood is needed. The
blood is prevented from clotting by use of a heparinized
syringe, since clumping of the blood
cells precludes culturing (the proportion of heparin
to blood is about 1: 20). Peripheral blood
or isolated lymphocytes are placed in a vessel
with culture medium. The cells are generally
stimulated with phytohemagglutinin, a protein
from plants that unspecifically stimulates
lymphocytes to divide. The culture requires
about 72 hours at 37!C for cells to divide.
Lymphocyte cultures are suspension cultures;
i. e., the cells divide in culture medium without
attaching to the culture vessel. Cell division is
arrested and the culture is terminated by
adding a suitable concentration of a colchicine
derivative (colcemid) two hours prior to
harvest. Colcemid interrupts mitosis during
metaphase, so that a relative enrichment of
cells in metaphase results.
Cell preparation is carried out as follows:
the
culture solution is centrifuged; the cell sediment
is placed in a hypo-osmolar KCI solution
(0.075 molar), incubated for about 20 minutes,
and centrifuged again. The resulting cell sediment
is placed in fixative. The fixing solution is
a mixture of methyl alcohol and glacial acetic
acid in a ratio of 3:1. Usually the fixative is
changed two to three times with subsequent
centrifugation. After that, the fixed cells are
taken up in a pipette and dropped onto a slide.
The preparation is stained, and the slide is
covered with a cover glass.
culture solution is centrifuged; the cell sediment
is placed in a hypo-osmolar KCI solution
(0.075 molar), incubated for about 20 minutes,
and centrifuged again. The resulting cell sediment
is placed in fixative. The fixing solution is
a mixture of methyl alcohol and glacial acetic
acid in a ratio of 3:1. Usually the fixative is
changed two to three times with subsequent
centrifugation. After that, the fixed cells are
taken up in a pipette and dropped onto a slide.
The preparation is stained, and the slide is
covered with a cover glass.
At this point the cells are ready for analysis.
Suitable metaphases are located under the microscope
with about 100x magnification and
are subsequently examined at about 1250x
magnification. During direct analysis with the
microscope, the number of chromosomes and
the presence or absence of all chromosomes
and recognizable chromosome segments are
noted. Since the preparation procedure itself
may induce deviations from the normal chromosome
number or structure in some cells,
more than one cell must be analyzed. Depending
on the purpose of the analysis, between 5
and 100 metaphases (usually 10–15) are examined.
Some of the metaphases are photographed
with the microscope and can subsequently
be cut out of the photograph (karyotyping).
In thisway a karyotype can be obtained
from the photograph of a metaphase. The time
needed for a chromosome analysis varies depending
on the problem, but is usually 3–4
hours. Analysis and karyotyping time can be
shortened by computer procedures.
with about 100x magnification and
are subsequently examined at about 1250x
magnification. During direct analysis with the
microscope, the number of chromosomes and
the presence or absence of all chromosomes
and recognizable chromosome segments are
noted. Since the preparation procedure itself
may induce deviations from the normal chromosome
number or structure in some cells,
more than one cell must be analyzed. Depending
on the purpose of the analysis, between 5
and 100 metaphases (usually 10–15) are examined.
Some of the metaphases are photographed
with the microscope and can subsequently
be cut out of the photograph (karyotyping).
In thisway a karyotype can be obtained
from the photograph of a metaphase. The time
needed for a chromosome analysis varies depending
on the problem, but is usually 3–4
hours. Analysis and karyotyping time can be
shortened by computer procedures.
In Situ Hybridization in Metaphase and Interphase
In situ hybridization refers to procedures that
demonstrate DNA sequences directly on chromosome
preparations (in situ). Since resolution
is relatively good (about 12 x 107 base pairs), the
exact regional localization of a sequence on its
corresponding chromosome can be determined.
demonstrate DNA sequences directly on chromosome
preparations (in situ). Since resolution
is relatively good (about 12 x 107 base pairs), the
exact regional localization of a sequence on its
corresponding chromosome can be determined.
Principle of in situ hybridization
Cells in metaphase or interphase are fixed on a
slide and denatured to change the doublestranded
DNA (1 a) into single-stranded DNA
(2). The metaphase or interphase preparation is
then hybridized (3) with DNA sequences that
are complementary to the region of interest and
that have been labeledwith biotin (1 b). The hybridization
site is made visible by means of a
primary antibody against biotin; this antibody
is bound to a fluorochrome (4), e. g., fluorescein
isothiocyanate (FITC). Since the primary signal
is quite weak, a secondary antibody (e. g.,
avidin) bound to biotin is attached (5). A further
primary antibody can then be attached to the
secondary antibody (6). This amplifies the signal,
which can then be demonstrated by bright
fluorescence under the light microscope.
slide and denatured to change the doublestranded
DNA (1 a) into single-stranded DNA
(2). The metaphase or interphase preparation is
then hybridized (3) with DNA sequences that
are complementary to the region of interest and
that have been labeledwith biotin (1 b). The hybridization
site is made visible by means of a
primary antibody against biotin; this antibody
is bound to a fluorochrome (4), e. g., fluorescein
isothiocyanate (FITC). Since the primary signal
is quite weak, a secondary antibody (e. g.,
avidin) bound to biotin is attached (5). A further
primary antibody can then be attached to the
secondary antibody (6). This amplifies the signal,
which can then be demonstrated by bright
fluorescence under the light microscope.
Demonstration of the Philadelphia translocation in chronic myeloid leukemia (CML)
The Philadelphia translocation (1) in chronic
myeloid leukemia (CML, see p. 332) can be demonstrate
in metaphase (2) and in interphase (3)
by means of in situ hybridization. When a probe
for the BCR gene is used in interphase, the normal
signal consists of two fluorescing dots, one
dot on each chromosome 22. (On good preparations
of metaphase chromosomes, one dot is
seen over each chromatid and appears as a
double dot on a chromosome.) When the probe
includes the breakpoint of the translocation,
three signals are visible: the largest over the
normal chromosome 22, a small one over the
BCR sequences remaining in the distal long arm
of a chromosome 22 (22q), and another small
one over the sequences translocated to the distal
long arm of chromosome 9.
myeloid leukemia (CML, see p. 332) can be demonstrate
in metaphase (2) and in interphase (3)
by means of in situ hybridization. When a probe
for the BCR gene is used in interphase, the normal
signal consists of two fluorescing dots, one
dot on each chromosome 22. (On good preparations
of metaphase chromosomes, one dot is
seen over each chromatid and appears as a
double dot on a chromosome.) When the probe
includes the breakpoint of the translocation,
three signals are visible: the largest over the
normal chromosome 22, a small one over the
BCR sequences remaining in the distal long arm
of a chromosome 22 (22q), and another small
one over the sequences translocated to the distal
long arm of chromosome 9.
Translocation 4;8
This preparation shows the translocation of
part of the long arm of a chromosome 8 to the
short arm of a chromosome 4 in a patient with
Langer–Giedion syndrome. The hybridization
was done with a 170-kb YAC (yeast artifical
chromosome) that spans the breakpoint of the
translocation in the 8q24 region. Three fluorescent
signals result: over the normal chromosome
8, over the part of 8q24 translocated to
chromosome 4, and over the sequences remaining
on chromosome 8. Chromosomes 4 and 8
were hybridizedwith alphoid probes, which are
specific for the centromere region.
part of the long arm of a chromosome 8 to the
short arm of a chromosome 4 in a patient with
Langer–Giedion syndrome. The hybridization
was done with a 170-kb YAC (yeast artifical
chromosome) that spans the breakpoint of the
translocation in the 8q24 region. Three fluorescent
signals result: over the normal chromosome
8, over the part of 8q24 translocated to
chromosome 4, and over the sequences remaining
on chromosome 8. Chromosomes 4 and 8
were hybridizedwith alphoid probes, which are
specific for the centromere region.
Telomere sequences in metaphase chromosomes
This illustration shows part of a human
metaphase chromosome after in situ hybridization
with telomere sequences (see p. 180). Each
chromosome shows four signals, one over each
end (telomere) of each chromatid, because the
telomeric sequences are the same for all chromosomes.
metaphase chromosome after in situ hybridization
with telomere sequences (see p. 180). Each
chromosome shows four signals, one over each
end (telomere) of each chromatid, because the
telomeric sequences are the same for all chromosomes.
Specific Metaphase Chromosome Identification
The unambiguous identification of each chromosome
is prerequisite to defining structural
alterations associated with chromosomal imbalance.
With banding pattern analysis, chromosomal
resolution is limited by the size of the
recognizable bands and the relative similarity
of bands of different chromosomes. The smallest
chromosomal region detectable by light microscopy
in banded metaphase preparations is
about 5–10 million base pairs (5–10Mb). Such
a segment could harbor 10–50 genes.
The methods utilize individual differences of
DNA sequences of each chromosome and
special techniques to induce different color images
of each chromosome pair (”chromosome
painting”). The DNA of metaphase chromosomes
is first denatured (made singlestranded)
and then hybridized on a slide in situ
toDNA probes that make up a large collection of
DNA fragments from one particular chromosome.
is prerequisite to defining structural
alterations associated with chromosomal imbalance.
With banding pattern analysis, chromosomal
resolution is limited by the size of the
recognizable bands and the relative similarity
of bands of different chromosomes. The smallest
chromosomal region detectable by light microscopy
in banded metaphase preparations is
about 5–10 million base pairs (5–10Mb). Such
a segment could harbor 10–50 genes.
The methods utilize individual differences of
DNA sequences of each chromosome and
special techniques to induce different color images
of each chromosome pair (”chromosome
painting”). The DNA of metaphase chromosomes
is first denatured (made singlestranded)
and then hybridized on a slide in situ
toDNA probes that make up a large collection of
DNA fragments from one particular chromosome.
DNA fragments
DNA fragments from one particular chromosome.
Probes for all 24 human chromosomes
are hybridized to the metaphase. Two approaches
have proved particularly useful: multiplex
fluorescence in situ hybridization (MFISH,
Speicher et al., 1996) and spectral karyotyping
(SKY, Schröck et al., 1996). Other approaches
and modifications exist; for example,
the use of artificially extended DNA or chromatin
fibers. In comparative genome hybridization
(CGH), the quantitative difference in allelic
sequences on homologous chromosomes is
compared to detect deletions or duplications
and amplifications.
Probes for all 24 human chromosomes
are hybridized to the metaphase. Two approaches
have proved particularly useful: multiplex
fluorescence in situ hybridization (MFISH,
Speicher et al., 1996) and spectral karyotyping
(SKY, Schröck et al., 1996). Other approaches
and modifications exist; for example,
the use of artificially extended DNA or chromatin
fibers. In comparative genome hybridization
(CGH), the quantitative difference in allelic
sequences on homologous chromosomes is
compared to detect deletions or duplications
and amplifications.
Multiplex FISH
With this approach (M-FISH), sets of chromosome-
specific DNA probes, each labeledwith its
own combination of DNA-binding fluorescent
dyes, are hybridized to metaphase chromosomes.
For each chromosome type, a specific
multicolor bar code is constructed by using
different YAC clones (yeast artificial chromosomes)
containing the DNA probes to be applied.
Only five different fluorophores are necessary
for image analysis by epifluorescence
microscopy using a charge-coupled device
(CCD) camera to generate a composite image of
each chromosome in a pseudocolor visualized
by appropriate software. The karyotype
generated in this way is composed of the 22
pairs of autosomes and the X and the Y each in a
different color.
specific DNA probes, each labeledwith its
own combination of DNA-binding fluorescent
dyes, are hybridized to metaphase chromosomes.
For each chromosome type, a specific
multicolor bar code is constructed by using
different YAC clones (yeast artificial chromosomes)
containing the DNA probes to be applied.
Only five different fluorophores are necessary
for image analysis by epifluorescence
microscopy using a charge-coupled device
(CCD) camera to generate a composite image of
each chromosome in a pseudocolor visualized
by appropriate software. The karyotype
generated in this way is composed of the 22
pairs of autosomes and the X and the Y each in a
different color.
Spectral karyotyping
Spectral karyotyping (SKY) combines Fourier
spectroscopy, CCD imaging, and optical microscopy.
The emission spectra of all points in the
sample aremeasured simultaneously in the visible
and near-infrared spectral range. Twentyfour
combinatorially labeled chromosome
paint probes, one specific to each chromosome
type, are hybridized to a metaphase after DNA
denaturation. The emission spectra of the individual
combinations of fluorophores are converted
to a spectrum of different visible display
colors by assigning blue, green, and red colors
to specific spectral ranges of fluorescent
wavelengths. The spectral karyotype is composed
of a specific false color for each chromosome
type. Spectral karyotyping has a wide
range of diagnostic applications in the analysis
of constitutional structural chromosome aberrations
and cancer cytogenetics.
spectroscopy, CCD imaging, and optical microscopy.
The emission spectra of all points in the
sample aremeasured simultaneously in the visible
and near-infrared spectral range. Twentyfour
combinatorially labeled chromosome
paint probes, one specific to each chromosome
type, are hybridized to a metaphase after DNA
denaturation. The emission spectra of the individual
combinations of fluorophores are converted
to a spectrum of different visible display
colors by assigning blue, green, and red colors
to specific spectral ranges of fluorescent
wavelengths. The spectral karyotype is composed
of a specific false color for each chromosome
type. Spectral karyotyping has a wide
range of diagnostic applications in the analysis
of constitutional structural chromosome aberrations
and cancer cytogenetics.
Numerical Chromosome Aberrations
Deviation from the normal chromosome number
in a single pair of chromosomes is referred
to as aneuploidy. In humans, numerical chromosome
aberrations occur in about 1 in 400
newborns. An abnormality of the number of
chromosomes occurs as a result of their abnormal
distribution (nondisjunction) during meiosis
I or II (meiotic nondisjunction). With meiotic
nondisjunction, the aberration occurs in all
cells of a resulting organism. Nondisjunction in
meiosis I and meiosis II can be differentiated
(see pp. 116). Abnormal chromosomal distribution
during mitosis leads to an aberration in
only a proportion of the cells (chromosomal
mosaicism).
in a single pair of chromosomes is referred
to as aneuploidy. In humans, numerical chromosome
aberrations occur in about 1 in 400
newborns. An abnormality of the number of
chromosomes occurs as a result of their abnormal
distribution (nondisjunction) during meiosis
I or II (meiotic nondisjunction). With meiotic
nondisjunction, the aberration occurs in all
cells of a resulting organism. Nondisjunction in
meiosis I and meiosis II can be differentiated
(see pp. 116). Abnormal chromosomal distribution
during mitosis leads to an aberration in
only a proportion of the cells (chromosomal
mosaicism).
Triploidy
Triploidy refers to a deviation from the normal
number of chromosomes in which each chromosome
is present threefold instead of twofold.
With tetraploidy, four copies of each chromosome
are present. Triploidy arises when an abnormal
oocyte with a double (46,XX) chromosome
complement instead of a haploid complement
(23,X) is formed. After fertilization by a
normal spermatocyte, triploidy (69,XXX or
69,XXY) of maternal origin arises. In this case,
two of the three complete sets of chromosomes
arematernal. Triploidy may also arise as a result
of abnormalities during spermatogenesis, resulting
in an abnormal spermatozoon that does
not contain the normal haploid chromosome
complement, but rather the diploid (46,XY). In
this case, the triploidy (69,XXY) is of paternal
origin (see p. 402). A further cause of triploidy is
dispermy, or fertilization of a normal egg by two
normal sperm.
number of chromosomes in which each chromosome
is present threefold instead of twofold.
With tetraploidy, four copies of each chromosome
are present. Triploidy arises when an abnormal
oocyte with a double (46,XX) chromosome
complement instead of a haploid complement
(23,X) is formed. After fertilization by a
normal spermatocyte, triploidy (69,XXX or
69,XXY) of maternal origin arises. In this case,
two of the three complete sets of chromosomes
arematernal. Triploidy may also arise as a result
of abnormalities during spermatogenesis, resulting
in an abnormal spermatozoon that does
not contain the normal haploid chromosome
complement, but rather the diploid (46,XY). In
this case, the triploidy (69,XXY) is of paternal
origin (see p. 402). A further cause of triploidy is
dispermy, or fertilization of a normal egg by two
normal sperm.
Aneuploidy
In a trisomy (1), only one of the chromosomes is
present threefold; all other chromosome pairs
are normal. Rarely, two different trisomies
occur, for two different chromosomes (double
aneuploidy). If one chromosome of a pair is
missing, it is referred to as monosomy (2).
present threefold; all other chromosome pairs
are normal. Rarely, two different trisomies
occur, for two different chromosomes (double
aneuploidy). If one chromosome of a pair is
missing, it is referred to as monosomy (2).
Origin of trisomy and monosomy
The result of normal meiosis , consisting
of two cell divisions (not shown here), is
a normal distribution and a haploid chromosome
complement. With abnormal distribution
(nondisjunction either in meiosis I or inmeiosis
II), one gamete is formed with an additional
chromosome, whereas the other is missing a
chromosome. After fertilization, the respective
zygote contains either three copies of one chromosome
(trisomy) or only a single chromosome
of a pair (monosomy). Abnormal chromosome
distribution can occur during oogenesis (maternal
nondisjunction) or during spermatogenesis
(paternal nondisjunction).
of two cell divisions (not shown here), is
a normal distribution and a haploid chromosome
complement. With abnormal distribution
(nondisjunction either in meiosis I or inmeiosis
II), one gamete is formed with an additional
chromosome, whereas the other is missing a
chromosome. After fertilization, the respective
zygote contains either three copies of one chromosome
(trisomy) or only a single chromosome
of a pair (monosomy). Abnormal chromosome
distribution can occur during oogenesis (maternal
nondisjunction) or during spermatogenesis
(paternal nondisjunction).
Abnormalities of chromosome number in humans
In humans, the following autosomal trisomies
may occur in liveborn infants: trisomy 13with a
frequency of 1 in 5000 newborns; trisomy 18
with 1 in 3000; and trisomy 21 with about 1 in
650 newborns (1). Additional X or Y chromosomes
occur in about 1 in 800 newborns, much
more frequently than the autosomal trisomies
(2). But unlike the autosomal trisomies, they
usually do not lead to defined clinical pictures.
Triple X (47,XXX) or an additional Y chromosome
(47,XYY) are generally not clinically apparent.
On the other hand, monosomy X (3)
leads to the clinical picture of Turner syndrome
may occur in liveborn infants: trisomy 13with a
frequency of 1 in 5000 newborns; trisomy 18
with 1 in 3000; and trisomy 21 with about 1 in
650 newborns (1). Additional X or Y chromosomes
occur in about 1 in 800 newborns, much
more frequently than the autosomal trisomies
(2). But unlike the autosomal trisomies, they
usually do not lead to defined clinical pictures.
Triple X (47,XXX) or an additional Y chromosome
(47,XYY) are generally not clinically apparent.
On the other hand, monosomy X (3)
leads to the clinical picture of Turner syndrome
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