T H E AMERICAN JOURNAL OF CLINICAL PATHOLOGY
Vol. 39, N o . 1, p p . 3-37
.liinuary, 1963
C o p y r i g h t © 1003 b y T h e Williams & Wilkins C o .
Printed in
U.S.A.
CYTOGENETICS
REVIEW OF RECENT ADVANCES IN A N E W FIELD OF CLINICAL PATHOLOGY
ROBERT R. EGGEN, M.D.
Department of Pathology, Mercy Hospital, Son Diego, California
Cytogenetics, one of the most intriguing
of the basic sciences, has recently come to
be of expanding clinical importance. Cytogenetics correlates microscopically discernible structural and numerical abnormalities
of chromosomes with the clinical changes
associated with them. More and more requests for cytogenetic studies will reach the
clinical laboratory as interest in the field
grows.
In the few years during which convenient
cytogenetic technics have been applied to
human disease, a sizeable body of literature
has accumulated, chiefly in the British
journals. Review articles'08' 183 are now appearing in the American literature, chiefly
in specialty journals. Inasmuch as the laboratory aspects of cytogenetics will ultimately
become the responsibility of the clinical
pathologist, a review of cytogenetic principles, current technics, and clinicopathologic
correlations should be of interest to readers
of this journal.
were first described in cats by Barr and
Bertram in 194913 and were later demonstrated in human skin biopsies.141 In 1954,
Davidson and Smith54 found similar nuclear
sex differences in human polymorphonuclear
leukocytes. The development of the buccal
smear technic,142 by circumventing the inconvenience of skin biopsy, made the Bantest a readily accepted laboratory procedure.
In vitro technics that permit visualization
of the chromosomes themselves are of more
recent origin. The normal human chromosome number was thought to be 48 until
Tjio and Levari in 1956192 indicated that
the normal (euploid) complement was, in
truth, 48 chromosomes. Their outstanding
work was soon confirmed by others, 42 ' 70 who
used the improved technics that Tjio and
Levan had described.
Unfortunately, the misconception that
visible chromosomal abnormalities in man
were unlikely to occur in viable persons (if,
indeed, they occurred at all) remained prevalent. It was, therefore, assumed that examination of chromosomes would offer little
of practical clinical value. This misconception was dispelled in 1959 by Lejeune and
associates,124 who demonstrated a constant
chromosomal, abnormality in mongolism.
Since then, many reports of clinically significant chromosomal anomalies have been published, and the importance of cytogenetics
to the clinician is now well established.
HISTORICAL CONSIDERATIONS
Cytogenetic technics have been in use
for more than 40 years, ever since Bridges29
first correlated structural chromosomal abnormalities with changes in the physical
characteristics (phenotype) of Drosbphila.
Until recently, however, technical difficulties
inherent in the study of mammalian tissues
have hindered application of this science
to man.
Cytogenetic technics applicable to man
include chromosome analysis per se and the
better known Barr chromatin test. The Barr
test is used to demonstrate sex differences
in nuclear morphology. These differences
NORMAL MECHAXISMS
Thorough acquaintance with the normal
mechanisms of cell division is essential to all
who attempt laboratory and clinical investigation in cytogenetics. To most physicians
the semantics of the field are, at best, only
vaguely familiar. A review of normal mitosis
and meiosis will help to give an understanding of the many errors that may occur in
human cell division.
Received, February 28, 1962; revision received,
July 5; accepted for publication, October 4.
Dr. Eggen is Associate Pathologist. His present
address is 3322 Congress Street, San Diego,
California.
3
EGGEN
Chromosomes per se are recognizable only
during active cell division. In the interphase
("resting") nuclei that predominate in tissue sections, genetic material is represented
by irregularly dispersed strands and clumps
of nuclear chromatin.
The discrete chromosomes seen in dividing
nuclei are long double helices of polymerized
DNA (deoxyribose nucleic acid) 20 ' 1202 united
with varying amounts of polypeptide or
incomplete protein. In intermitotic nuclei,
the DNA polymers are duplicated by mechanisms as yet unknown. The complex mechanisms of cell division serve to separate these
duplicated DNA polymers and to distribute
them to daughter cells as new chromosomes.
The DNA helix has a "backbone" of
ribose-phosphate units that bears nucleic
acid side chains. The spatial orientation of
the 4 nucleic acids of DNA (cytosine, thymine, guanine, and adenine) provides a
"code" by which genetic information is
transmitted from generation to generation.
In addition, nuclear DNA mediates the
functional activity of the cell by transmitting its information via cytoplasmic "messenger" units of RNA (ribonucleic acid).85
These units pick up amino acids and line
up (possibly along other cytoplasmic RNA
molecules) to form polypeptides and proteins, many of which function as cellular
enzymes.
Thus, the concept that the gene is a separate physical entity has been superseded by
the DNA "code" concept of inheritance.
Recent investigation of bacterial RNA162
suggests, for instance, that the transport of
phenylalanine depends upon the sequential
arrangement of 3 uracil bases.53 In all likelihood, "information" regions of the helix are
separated from one another by "nonsense"
regions having no useful genetic information.56 Such findings have modified only the
concept of the gene's physical structure;
each information area (gene) is still believed
to govern a specific phenotypic feature.
The mechanisms of cell division that are
important to cytogeneticists are mitosis and
meiosis. Mitosis, cell division in somatic cells,
produces 2 new (daughter) cells, each of
which contains the same (diploid or 2n) number of chromosomes as did the parent cell.
Vol 39
Gametes are produced by meiosis, in which
each parent germ cell (2n) forms 4 haploid
(n) daughter cells.
Mitosis
Mitosis is divided into 5 phases,204 the
first being interphase. To refer to interphase
nuclei as "resting" is semantically incorrect,
inasmuch as it is within these nuclei that
the most significant part of cell division—
namely, duplication of the DNA helix—
occurs.
Chromosomes become recognizable during
the second phase, prophase, as double strands
of densely stained chromatin united at a
pale or unstained area, the centromere. The
centromere may be at or near the midpoint
of the double strands (metacentric), near
the ends (acrocentric), or in an eccentric
position (submetacentric) that demarcates
the strands into readily apparent long and
short arms. Terminal (telocentric) centromeres are not seen in normal human material. Each of the strands is a chromatid;
the entire complex (2 chromatids plus centromere) is a single chromosome (Fig. 1).
During prophase, lysis of the nuclear
membrane begins; the cell wall remains intact throughout mitosis. Two centrioles are
visible at this time as they begin to migrate
to the poles of the cell, where they ultimately serve as points of attachment for the
spindle body. Also during prophase, the
acrocentric chromosomes are intimately associated with the nucleolus and are thought
to function as nucleolus organizers in the
human cell.150 They are subject to significant mechanical stresses, becoming stretched
and elongated to a much greater degree than
other chromosomes. The relation between
them and the nucleolus, apparently peculiar
to the acrocentric chromosomes, may be
responsible for the fact that anomalies affect them more often than the other kinds.
During the third phase of mitosis, metaphase, lysis of the nuclear membrane is completed. At the same time, the chromatids
become shorter and plumper (the DNA
helix contracts) and the chromosomes assume the " X " and "wishbone" configurations in which they are most often depicted.
As metaphase progresses, the 46 chromo-
MITOSIS
F I G . 1 (upper). Stages of mitosis. The chromatin strands of the interphase nucleus (a)
condense to form chromonemata in early prophase (b). In late prophase (c), as the nuclear
membrane lyses, chromosomes form and then split longitudinally into paired chromatids
united by a centromere. The chromosomes migrate to the equatorial plate during metaphase ((/), and t h e spindle body forms. T h e centromere splits and the chromatids migrate
to the opposite poles of the cell and the nuclear membrane re-forms in late metaphase (e).
Two new nuclei, with dispersed chromatin, form in telophase (/). Only nuclear changes
arc illustrated; the cell wall is not drawn.
F I G . 2 (lower). Stages of meiosis. Homologous chromosomes are illustrated. During the
first meiotic division (a-d), nuclear chromatin condenses and forms chromonemata. (a),
which become closely apposed in homologous pairs, bivalents, (6) along the equatorial
plate. Jn late prophase (c), the chromosomes split longitudinally, and the 2 pairs of chromatids produced form a tetrad. As the centermost chromatids contact each other, crossingover occurs and a mutually repulsive force develops between the chromosomes. Whole
chromosomes migrate to the cell poles in metaphase I (d).
During the second meiotic division (e-h), the centromeres split (/), and the chromatids migrate to opposite poles of the cell ({/). By the end of meiosis (A), each germ cell has
produced 4 haploid daughter cells. Only nuclear reactions are illustrated; the cell wall
is not drawn.
5
6
EGGEN
somes form an irregular disk at the center
of the cell (the equatorial plate), apparently
in a random fashion. Concomitant with
formation of the equatorial plate, the nuclear
sap condenses to form the spindle apparatus,
a series of sticky, tenacious threads attached
at one end to a chromosome and at the other
to a centriole. The chromosomal point of
attachment is the centromere, and some of
the smaller chromosomes may become completely enveloped by the spindle body. Metaphase figures are the most convenient for
chromosome studies.
In the fourth phase, anaphase, the centromeres split and the separated chromatids
migrate to opposite poles of the cell. After
migration of the chromatids, the spindle
body disappears and the nuclear membrane
begins to re-form.
In the fifth and final phase, telophase, reconstitution of the nuclear membrane is
completed and the genetic material of the
chromatids (which, by now, are regarded as
new chromosomes) becomes dispersed
through the nuclear sap as nuclear chromatin. A temporary binucleate cell is produced and is then converted into 2 separate
daughter cells as an intervening cell wall
develops between the 2 nuclei. Complete
segregation into 2 discrete cells marks the
end of telophase.
The net result of a mitotic cell division is
the production of 2 daughter cells, each of
which has the same number of chromosomes
as the parent cell had. Each chromosome of
the parent cell is halved, and 1 half of each
chromosome is given to each daughter cell.
The processes of chromosomal splitting and
polar migration are referred to collectively
as disjunction. Errors of this mechanism,
of which nondisjunction (Fig. 5) seems to be
most frequent, account for many of the abnormalities of chromosome number (aneuploidy).
Meiosis
Meiosis (Fig. 2) is a specialized form of
cell division, unique to germ cells, that occurs during gametogenesis. The daughter
cells (gametes) of a meiotic division have
only half as many chromosomes (haploid) as
the parent cell from which they arise. Were
Vol. 39
it not for this specialized form of cell division
in gametogenesis, the chromosome content
of body cells would double with each succeeding generation.
In addition to giving the daughter cells
only half as many chromosomes (23 rather
than 4.6, in man), meiosis differs from mitosis
in other important respects: (1) the DNA
helix is duplicated only once, although 2 cell
divisions occur; (2) at the equatorial plate
chromosomes arrange themselves in homologous pairs (synapsis) rather than at random as in mitosis; and (3) there is an exchange of genetic material between
homologous chromosomes (crossing-over) at
points of contact (chiasmala) between the
chromatids.
A meiotic division embraces 2 successive
divisions. The first one, reductional division,
produces 2 daughter cells that are haploid.
In the second meiotic division, equalional
division, the chromosomes of these haploid
daughter cells divide at their centromeres
and produce a second generation of daughter
cells that are also haploid. Thus, each parent
(germ) cell ultimately produces 4 haploid
daughter cells (gametes).
Each meiotic division has 5 phases similar
to those of a mitotic division. Prophase I
(prophase of the first meiotic division) itself
has 5 recognizable stages. Lysis of the nuclear membrane takes place during prophase
I, concomitant with the chromosomal
changes. During leptonema, the first of the
5 stages of prophase I, nuclear chromatin
condenses into elongated stringlike bodies,
chromonemata, which further condense to
form elongated but recognizable chromosomes. In zygonema, the chromonemata migrate to the equatorial plate and become
arranged in closely apposed homologous
pairs (synapsis), which are referred to as
bivalents. In pachynema, the members of the
bivalent become shorter and plumper as the
DNA helices continue to contract. During
diplonema, each of the chromosomes of the
bivalent splits in its long axis to form 2
chromatids that remain united at the centromere. The resulting complex of 4 closely
apposed chromatids is a tetrad. At chiasmata, the 2 centermost chromatids come
into intimate contact and an actual ex-
Jan. 1963
CYTOGENETICS—A REVIEW
change of genetic material takes place between them (crossing-over). During the
fifth stage, diakinesis, concomitant with the
formation of chiasmata, a mutually repulsive
force develops between the paired chromosomes, and encourages migration of the
chromosomes to opposite poles of the cell.
The remaining phases of the first meiotic
division are similar to those of a mitotic cell
division. During telophase, the nuclear membrane re-forms, and the binucleate cell that
results is divided into 2 daughter cells by
the growth of an intervening cell wall. At
the conclusion of the first meiotic division,
the daughter cells are haploid, each containing 1 member of each of the chromosome
pairs of the parent germ cell.
The second meiotic division may begin
immediately after the first, or there may be
a period of inactivity (interkinesis) of varying duration. During prophase II, the chromosomes (paired chromatids) of the daughter cells form an equatorial plate. In
metaphase II, the centromere that unites
the chromatids splits and the chromatids
migrate to opposite poles of the cell (anaphase II). Nuclear membranes re-form and,
by the end of telophase II, each of the daughter cells of the first meiotic division has
produced 2 "second generation" daughter
cells (gametes).
One of the striking differences between
mitosis and meiosis is noted during the first
meiotic division in that an entire chromosome migrates to a cell pole without separation of the chromatids. In mitosis, entire
chromosomes do not migrate in this fashion;
the centromere splits and the chromatids
migrate to the poles of the cell. Separation
of chromatids in meiosis does not occur until
the second meiotic division.
Meiotic disjunction occurs twice: first,
with separation of the chromosome pairs in
anaphase I, and later, with separation and
migration of the chromatids in metaphase II
and anaphase II. Meiotic disjunction is
prone to many accidents, of which nondisjunction (Fig. 4.) is the most common. Determining which meiotic disjunction is more
prone to accident is difficult. Most descriptions seem to favor the second one although at least 1 author146 has implicated
7
nondisjunction in the first meiotic division
as a cause of aneuploidy. Meiotic chromosomes are also susceptible to fracturing. Accidents of meiosis have been implicated in
many of the known chromosomal defects.
LABORATORY METHODS
Until recently, the study of mammalian
chromosomes has been hindered by technical
difficulties engendered by the spindle apparatus. This gluey, multithreaded structure is attached to, and often envelops,
metaphase chromosomes and causes a degree of tangling and piling that makes accurate examination virtually impossible.
This impediment can be overcome by
treating cell cultures with colchicine. Colchicine arrests cell division at metaphase by
inhibiting formation of the spindle apparatus,45 and there is a consequent accumulation
of easily spread metaphase figures. Many
current culture methods utilize colchicine,
although some investigators121 have found
that the anticoagulant EDTA has an action
similar to that of colchicine.
Any tissue in which there are cells capable
of dividing in culture mediums may be used
for chromosome analysis. The most convenient tissues for cytogenetic studies are
blood, bone marrow, skin, and fascia lata.
In a complete cytogenetic study, at least 2
different tissues should be cultured, inasmuch as some instances of mosaicism may
escape notice if only 1 tissue is used. (Differences in karyotype may be found even between blood and bone marrow cultures.125)
Adherence to a sterile technic is, of course,
mandatory.
Technics used for the examination of
blood, bone marrow, and solid tissues vary
considerably. The following remarks outline
some of the principles upon which culture
methods are based. Detailed methodology
is presented in the Appendix.
Cultures of peripheral blood leukocytes
present the dual advantages of ease of sampling and comparative rapidity of growth.
Phytohemagglutinin, used by most investigators in preparing blood for culture, has 2
valuable properties. It enhances sedimentation of erythrocytes, providing a supernatant fluid rich in leukocytes, and, equally
8
Vol. 89
EGGEN
important, it enhances the mitotic activity
of leukocytes. The latter action, originally
attributed to contaminating plant growth
hormone,38 has since been found to be an
inherent property of phytohemagglutinin
itself.49 Culture times of 2 to 3 days usually
yield cell suspensions of good quality for
examination.
Methods for the culture of bone marrow
are, in many respects, similar to those used
for peripheral blood. The technical differences between them relate to the infinitely
greater cellularity of bone marrow. The
amount of material required is small, and
the use of phytohemagglutinin is rarely
necessary, inasmuch as the inherently high
mitotic activity of bone marrow minimizes
the need for mitogenic activity.
Solid tissues (skin, fascia) are less popular
sources of culture material. They are less
convenient to obtain and, because of the
relatively long culture times needed (usually
2 weeks) to obtain extensive cell layers for
study, require considerably more manipulation than do blood or bone marrow. Initial
culture utilizes tissue fragments 0.5 to 1.0
mm. in diameter, usually grown out as a
monolayer of cells on coverslips. Cell suspensions are prepared by treating the monolayers with trypsin.
Whatever the tissue selected for culture,
it is important to make the culture time as
brief as possible, consistent with obtaining
satisfactory preparations. Prolonged culture
may induce factitious alterations in chromosome patterns.
Final preparations of the cultured cells
are similar regardless of the source of the
material. Most workers use colchicine near
the end of the culture period in order to enhance the accumulation of metaphase figures. The cells are harvested and are treated
with hypotonic saline solution or citrate to
promote swelling of the cells and dispersion
of chromosomes.
Smears are prepared from the suspensions,
and further separation of chromosomes is
encouraged either by means of air-drying
or by gentle pressure on the coverslip of wet
mounted preparations. It has been demonstrated189 that air-drying leads to less chromosome loss than squashing, but tends to
TABLE 1
HYPOTHETICAL CHROMOSOME C O U N T S
Condition
Normal*
Number of
Chromosomes
Number of Cells
<44
44
45
46
47
48
>48
0
1
2
78
1
0
0
82
<44
44
45
46
47
48
>48
0
0
1
3
57
0
0
61
<44
44
45
46
47
48
>48
0
1
22
32
1
0
0
56
Total
Trisomy!
Total
Mosaicismt
Total
* The modal count of 46 is typical (diploid).
As indicated, individual variations less t h a n the
mode are more common t h a n those greater t h a n
the mode; they presumably reflect technical loss
of chromosomes.
t A modal count of 47 is characteristic of
trisomy.
| A bimodal count of this type, with aneuploidy of approximately half of t h e cells inspected, is characteristic of mosaicism.
leave chromosomes "fuzzy" in microscopic
appearance.
Smears are stained, generally with orcein,
and examined under oil immersion. The
chromosomes are counted in as many cells
as manifest intact and well dispersed metaphase figures. Typical counts are exemplified
in Table 1. A modal count other than the
normal (46) indicates aneuploidy. Occasional
cells yielding nonmodal counts will be encountered in most preparations. Individual
variations are usually owing to technical fac-
Jan. 1963
CYTOGENETICS—A
tors and should not affect more than 3 per
cent of the cells examined.61
Photographs or camera lucida drawings
are made of well dispersed metaphase figures having a modal count. A final magnification of approximately 3000 is satisfactory.
Individual chromosomes are then arranged
in homologous pairs and in descending order
of length. The resulting figure is a karyotype;
diagrammatic presentations, such as Figure
3, are ideograms.
IDENTIFICATION AND N O M E N C L A T U R E
Fortunately, variations in nomenclature,
which often confuse the literature dealing
with a field as new as cytogenetics, were
anticipated and circumvented by early investigators. At a meeting held in Denver in
I960, a standard nomenclature was proposed and criteria for the identification of
individual chromosomes were tentatively established.22 The standard (Denver) nomenclature has been adopted by most workers
(Fig. 3).
Criteria used in the identification of chromosomes include length, relative position of
the centromere, and the ratio of long arm
length to short arm length. There are 7
major groups, which are designated (Denver
system) by the numbers of the chromosomes
they contain. Patau 159, 160 suggested letter
designations for the major groups, and these
are also used below.
Identifying features of the major chromosome groups are:
Group A (1 to 3)—large chromosomes
with median or nearly median centromeres
Group B (4 to 5)—large chromosomes
with submedian centromeres
Group C (G to 12 and X)—medium-sized
chromosomes with submedian centromeres
Group D (13 to 15)—medium-sized acrocentric chromosomes, which may bear
satellites on their short arms
Group E (16 to 18)—rather short chromosomes with median or submedian
centromeres
Group F (.1.9 to 20)—short chromosomes
with median centromeres
Group G (21 to 22 and Y)—very short
REVIEW
9
acrocentric chromosomes, which may
bear satellites on their short arms.
Chromosome identification in the laboratory is considerably more difficult than
casual inspection of the literature would
suggest. Although it is not especially difficult to assign a given chromosome to I of
the 7 major groups, accurate identification
of individual chromosomes is at best extremely difficult and, more often than not,
impossible. This point, we feel, has not received sufficient emphasis. Virtually all of
the karyotypes depicted in the literature,
by showing the chromosomes fully "identified" and labeled, lead one to believe that
individual chromosomes are being identified
routinely and without undue difficulty.
The criteria proposed for the identification of chromosomes are far from absolute.
Inspection of the data appended to the
Denver report22 will reveal a great deal of
overlapping. Additional criteria for the identification of individual chromosomes are
published from time to time, and the conflict among them emphasizes the hazards of
chromosome identification. For example,
the Y chromosome has been variously regarded as the longest of,73,1M the smallest
of,42 and the same size as other70 small acrocentric chromosomes (2.1 and 22). More
recently, reports 16 ' 207 have indicated that
the Y chromosome varies in size not only
from person to person but from cell to cell
within the same person. Criteria proposed
for the identification of the X chromosome51' 147' 15!'' 172 manifest a similar lack of
agreement.
It is the opinion of 'Patau,100 who has reported extensively on the subject of chromosome identification, that only the chromosomes of Groups A (I, 2, and 3) and E (16,
17, and .1.8) can be identified with reasonable
certainty in average preparations. If homologous chromosomes can be paired with confidence, then the members of Groups B (4.
and 5) and F (19 and 20) can be distinguished fairly accurately.
Accurate identification of individual
Group C chromosomes (6 to 12 and X) is
virtually impossible. Although it is usually
easy enough to distinguish the largest of the
group from the smallest, the variation in
10
Vol. 39
EGGEN
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6
7
13
14
8
15
9
10
16
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11
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17
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FEMALE
MALE
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if
xx
4
19
20
21
22
Y
F I G . 3 (upper). Ideogram of human chromosomes. T h e individual chromosomes are
designated according to the Denver system. T h e letter designations indicate the 7 major
groups. The Group C chromosomes manifest only minimal size variation between adjacent pairs, although the difference in length between the largest and smallest of the
group is fairly distinct (compare A' and IS in the ideogram). The sex cliromosomes appear
as members of Groups C (X chromosome) and G (Y chromosome). Note the satellites
depicted on some of the acrocentric chromosomes (Group D and Group G).
F I G . 4 (lower). Nondisjunction in meiosis. Nondisjunction in the second meiotic division is illustrated. A haploid daughter cell (a) from the first meiotic division contains
2 chromatids united by a centromere (i.e., 1 chromosome). If these chromatids fail to
separate, 2 abnormal gametes are produced (6). When these are fertilized, 4 types of
zygotes may be produced (c).
Jan.
1963
CYTOGENETICS—A REVIEW
size between adjacent pairs is minimal, and
arranging the group in an accurate sequence
of homologous pairs presents great difficulty.
In the Denver report the presence of
satellites on some of the acrocentric chromosomes (Groups D and G) was used as a differentiating feature. Satellites are technically
difficult to demonstrate because of their
minute size and fragility; however, they have
been found, at one time or another, attached
to each of the acrocentric chromosomes,69,70
although no one has yet reported all satellites present simultaneously. Hence, satellites are not a reliable differentiating feature
(except, perhaps, in the case of Chromosome
13, which is said to bear strikingly large
satellites.)156
The difficulty in identifying the sex chromosomes has been alluded to. In this regard,
it should be remembered that the determination of genetic sex can be made easily, even
when the identification of the X and Y
chromosomes per se is obscure. Normal females will have 16 Group C (XX) and 4
Group G chromosomes. Normal males will
have 15 Group C (X) and 5 Group G (Y)
chromosomes.
It is probably desirable, therefore, to accept the recommendation of Patau and to
refrain from identifying specific chromosomes unless it can be done with reasonable
scientific accuracy. This view is particularly
pertinent when chromosomal anomalies are
being reported; such anomalies should not
be attributed to a specific chromosome unless
the criteria for its identification are unequivocal.
B.
CLASSIFICATION OF CHROMOSOMAL,
ABNORMALITIES
Chromosomal abnormalities are those
either of number (aneuploidy) or of structure, occasionally of both. We believe that
the classification proposed below is sufficiently flexible to incorporate future findings
and that the distinction made between autosomal and sex chromosomal abnormalities
will prove clinically useful. Abnormalities
identified, or tentatively identified, to date,
are classified on this basis in Table 2.
A. Aneuploidy (abnormalities of number)
1. Isolated monosomy (2n = 45)—absence of
1 member of a chromosome pair; no other
C.
D.
E.
11
chromosomal abnormalities
a. Autosomal
b. Sex chromosomal
2. Isolated trisomy (2n = 47)—1 chromosome
appears in triplicate; no other chromosomal
abnormalities
a. Autosomal
b . Sex chromosomal
3. Isolated polysomy (2n = 48 or more)—1
chromosome is represented 4 or more times;
no other chromosomal abnormalities
a. Autosomal
b . Sex chromosomal
4. Complex aneuploidy—numerical variation
affecting 2 or more chromosomes; no associated structural abnormality
a. Autosomal
b. Combined autosomal and sex chromosomal
5. Aneuploid mosaics—aneuploidy of a portion, rather than of all, of the body cells
a. Autosomal
b. Sex chromosomal
Morphologic abnormalities
1. Translocations
a. Autosomal
b. Sex chromosomal
2. Deletions
a. Autosomal
b. Sex chromosomal
3. Duplications (partial trisomy)
a. Autosomal
b. Sex chromosomal
4. Isochromosomes
a. Autosomal
b. Sex chromosomal
5. Inversions
a. Autosomal
b . Sex chromosomal
6. R a r e and complex structural anomalies
a. R a r e anomalies involving a single
chromosome
b. Unrelated structural anomalies t h a t
affect 2 or more chromosomes simultaneously
Structurally anomalous aneuploids—structural change in 1 chromosome associated with
aneuploidy of another
a. Autosomal
b. Combined autosomal and sex chromosomal
Structurally anomalous mosaics—structural
anomalies affecting chromosomes in a portion,
rather than in all, of the body cells
a. Autosomal
b. Sex chromosomal
Clones—structural anomalies or aneuploidy
affecting a single line of cells (within 1 tissue)
t h a t is structurally and often physiologically
TABLE 2
CLASSIFICATION OF K N O W N CHROMOSOMAL
ABNORMALITIES
A. Aneuploidy
1. Isolated monosomy
a. Autosomal: no cases described
b. Sex chromosomal: XO—Turner's syndrome 7 ' '*• 157
2. Isolated trisomy
a. Autosomal
(1) ?Trisomy-3—complete sex reversal 157
(2) PGroup C trisomy—mental deficiency 171
(3) Group D trisomy—Group D trisomy syndrome 6 5 ' 129' 161 ' 190
(4) Group E trisomy—Group E trisomy syndrome 6 2 - 107' 145' 182
(5) Trisomy-19—asymptomatic 8 1
(6) Group F trisomy—heart disease 2 5
(7) Trisomy-21—sporadic mongolism 124
(8) Trisomy-22—?Sturge-Weber syndrome, 1 0 5 Pschizophrenia, 8 ' 19c Pbenign congenital myotonia 5 8
b. Sex chromosomal
(1) XXX—variable findings 113 ' 117
(2) XXY—Klinefelter's syndrome 7 1 ' ' "
(3) XYY— 1 8 ' 148' 173
3. Isolated polysomy
a. Autosomal: no cases described
b . Sex chromosmal
(1) X X X X "
(2) X X X Y 7 2 ' 131
(3) X X X X Y 2 . 8 »' 82 ' i«
(4) XXYY 3 3 ' ' " • 14s
4. Complex aneuploidy
a. Autosomal
(1) Monosomy-trisomy
(a) Trisomy-21, monosomy-16—mongolism 80
(b) Trisomy-21, monosomy-12—mongolism 80
(c) Trisomy-Group F , monosomy-Group G—heart disease 2 6
(2) Multiple trisomy: trisomy-8, trisomy-11, XXY 8 0
(3) Other forms: triploidy (3n = 69) 23 ' 24 ' 55 ' 139
b. Combined autosomal and sex chromosomal
(1) Trisomy-8, trisomy-11, XXY 8 0
(2) Trisomy-18, X X X 1 9 8
(3) Trisomy-21, XXY—mongolism and Klinefelter's syndrome 7 7 ' 101- u l ' 122
5. Aneuploid mosaics
a. Autosomal
(1) 4G/47-Group C trisomy 1 1 4
(2) 46/47-trisomy-21—mongolism 4 4 ' "• 92- 103' >51' 169
b . Sex chromosomal
(1) 45 XO/46 XX—chromatin positive gonadal dysgenesis 3 9 ' 89
(2) 45 XO/4G XY—amenorrhea, 1 9 - 114' 136. 209 hermaphroditism 1 0 9 ' 135
(3) 45 XO/47 XXX—gonadal dysgenesis 1 1 5
(4) 45 XO/47 XYY—amenorrhea 5 0 ' 114
(5) ?4G X X / 4 6 XY—hermaphroditism 2 0 3
(6) 4G X X / 4 7 XXY—Klinefelter's syndrome 1 1 5
(7) 4G XX/47 XXX—hermaphroditism 7 3 ' 90 ' 131
(8) 4G XY/47 XXY—Klinefelter's syndrome 8 ' 106 ' 131
(9) 48 X X X Y / 4 9 X X X X Y — m e n t a l deficiency 131
(10) 45 XO/46 X X / 4 7 XXX—chromatin positive gonadal dysgenesis 1 0 0
(11) ?45 XO/46 XY/47 XYY*—amenorrhea 114
(12) 45 XO/46 XY/47 XXY—mental deficiency 131
(13) 4G XY/47 X Y Y / 4 8 XXYY—mental deficiency 131
12
Jan.
196S
CYTOGENETICS—A REVIEW
TABLE
13
2—Concluded
B. Morphologic abnormalities
1. Translocations
a. Autosomal
(1) ?Group A/Group C "
(2) ?Long arm 2/2—Waldenstrom's macroglobulinemia 8 6
(3) 21/13—Marfan's syndrome 1 6 6
(4) 21/15—familial mongolism 3 6 . **• «• 81- "• »'• ' «
(5) 21/21—familial mongolism". '«• «»• 197
(0) 18/Group G—multiple anomalies 2 0 6
(7) 22/13—mental deficiency,' 97 heart disease 143
b. Sex chromosomal
(1) ?X/X—amenorrhea 6 0
(2) ?X/Y—complete sex reversal 157
(3) ?X/i) 60
(4) PY/21' 9 7
2. Deletions
a. Autosomal: no cases described
b. Sex chromosomal
(1) Deletion long arm X 9 0 . U 5
(2) Deletion short arm X 114
(3) Deletion Y47- ' " • »»
3. Duplications ("partial t r i s o m y " )
ii. Autosomal
(1) 17 or 18—Group E trisomy syndrome 1 4 5 . 162. ISI- 206
(2) 22—Sturge-Weber syndrome 1 6 2
b. Sex chromosomal: ?some isochromosome-X forms' 14
4. Isochromosomes
a. Autosomal: ?isochromosome-2—Waldenstrom's m a c r o g l o b u l i n e m i a " - " • 8 * 15S
b. Sex chromosomal: isochromosome-X 2 0 ' '">• U4
5. Inversions
a. Autosomal: Ppericentric inversion-21—mongolism 88
b. Sex chromosomal: no cases described
G. Rare and complex structural anomalies
a. Mare anomalies
(1) Ring autosomes—postirradiation 1 9 3
(2) Ring X—gonadal dysgenesis 127
(3) fsochromosome of deleted long arms of X 6 6
b. Complex anomalies: no cases reported
C. Structurally anomalous aneuploids
a. Autosomal: no cases described
b. Combined autosomal and sex chromosomal: 14/15 translocation with XXY—polydysspondj'lism 197
D . Structurally anomalous mosaics
a. Autosomal: no cases described
b. Sex chromosomal
(1) 45 XO/4G isochromosomes-X 20 - 126
(2) ?4(i XX/4G X-deleted x 9 °. 203
(3) 46 X X / 4 7 X X plus fragment 200
E . Clones
1. P h ' chromosome—chronic myelocytic leukemia 5 1 . 153
2. Ph' chromosome plus fragment—chronic myelocytic leukemia'
3. Extra, submetacentric—acute myelogenous leukemia 121
14
Vol. 89
EGGEN
independent of the rest of the body cells and
the remaining cells of the same tissue
ETIOLOGY OF CHROMOSOMAL
ABNORMALITIES
Factors associated with chromosomal
normalities with a frequency that can
be attributed to chance are summarized
low. The mechanisms involved in their
tion are unknown.
abnot
beac-
Maternal Age
In animals and in man, a high incidence
of congenital anomalies is related almost
linearly to advanced maternal age.67 This
relation, best documented for the sporadic
form of mongolism,34' 96,168 has been apparent to some degree in most human chromosomal anomalies (except Turner's syndrome). There is no similar correlation
between incidence and paternal age. The
likelihood that all of the ova that the human
female will produce are present at birth
implies that aging exerts some deleterious
effect that is spared spermatozoa because
of their continual production.
Radiation
Gonadal radiation in experimental animals will give rise to detectable mutations
in zygotes, but a similar causal relation in
man has not been convincingly demonstrated. Recent investigations by several
groups 30,46, 188, m have revealed that, in
blood cells at.least, exposure to radiation
results in transient production of chromosomal abnormalities, as does administration
of nitrogen mustard.48 The changes induced
have usually been of large enough magnitude to have been likely to cause nonviable
zygotes if they had been produced in germ
cells as well. Predictable relations between
radiation dosage and mutation rate have
not yet been discovered in man. Retrospective studies199 suggesting a relation between
maternal radiation and mongolism remain
unconfirmed.36
Chronic Disease
There are sporadic reports205 of chronic
disease in a mother associated with chromosomal abnormality in her offspring. The
increased incidence of abnormalities demonstrable in animals in these circumstances is
probably present in man, as well. Reports
to date are far too infrequent, however, to
rule out coincidence.
Genetic Influences
Chromosomal abnormalities have been
encountered in several members of the same
family25' M, m, n4, IOO, 197 a n d m a y a f f e c t a
different chromosome in each person. In
addition, several reports' 02,190 ' 19T have indicated that a parental chromosome abnormality predisposes the children to development of chromosomal anomalies (not
necessarily similar). Such relations suggest128
that there is a genetic locus that controls
normal cell division. A mutation at such a
locus could well induce an inheritable tendency to errors in germ cell division and consequent chromosomal abnormalities.
Mutagenic Viruses
Virus infection of a bacterium can alter
the genetic composition of the host bacterium. This phenomenon, most common in
lysogenic strains, is responsible for such
bacterial genetic alterations as exotoxin production by Corynebacterium diphtheriae.m
A^iral DNA is incorporated into the host
cell's chromosomal DNA, thereby imparting
to the host cell a new genetic constitution,
although not always producing detectable
phenotypic changes.
The phenomenon noted in animal tumors
(e.g., the rabbit papilloma) of a demonstrable
virus that is transmitted from cell generation
to cell generation suggests a similar relation.
As yet, however, there is no evidence that
viral DJSTA in these instances becomes incorporated into the host cell's chromosomes.
Human mutagenic viruses have not been
demonstrated to date.
MECHANICS OP CHROMOSOMAL
ABNORMALITIES
A disproportionate number of chromosomal abnormalities seems to affect the acrocentric chromosomes. This circumstance
may be a consequence of the specialized
functions of these chromosomes. FergusonSmith and Handmaker 69 have demonstrated
Jan.
1963
CYTOGENETICS—A REVIEW
that all of the acrocentrics may, at one time
or another, bear satellites on their short
arms. These chromosomes also reveal a
striking tendency to adhere to one another
during cell division.CL In addition, Ohno and
associates166 have observed that these chromosomes, which bear an intimate relation
to the nucleolus during cell division, apparently serve as the nucleolus organizers
in human cells. A similar relation has been
noted for Chromosome I.178
These chromosomes become much elongated and distorted as they are stretched
over the nucleolus. The combination of apposition and distortion undoubtedly predisposes to increased incidences of breakage
and of nondisjunction. The frequency of
anomalies involving the acrocentric chromosomes may be more apparent than real.
The acrocentrics carry relatively minute
amounts of genetic material on their short
arms,'97 and it is possible that losses of
genetic material of greater magnitude would
be lethal162 and so remain undetected.
Nondisjunction
Failure of a chromatid pair to separate
before migrating to the poles of the dividing
cell, referred to as nondisjunction (Figs. 4
and 5), produces aneuploid daughter cells.
The chromatids that migrate to the cell
pole still united function as 2 separate chromosomes when cell division is completed.
Premature disjunction and simple loss of a
chromosome through anaphase lagging are
other possible, but apparently less common,
causes of aneuploidy. Meiotic nondisjunction (Fig. 4), which is probably the most
common cause of aneuploidy, has predictable
effects on the genetic complement of the
affected gametes and on the zygotes derived from them.
Mitotic nondisjunction also occurs (Fig.
5), probably with a frequency at least equal
to that of meiotic nondisjunction. The results of mitotic nondisjunction are subject
to virtually infinite variation, altered as
they are by the viability of the nondisjunctive cells and by the number of normal cell
divisions that have preceded the nondisjunctive division. Two of the many possible
patterns are illustrated in Figure 5, .4 and B.
15
Mitotic nondisjunction occurring during
the first zygotic cell division (i.e., immediately after fertilization) yields I of 2 possible
genetic patterns. If both of the disjunctional
cells are viable and continue to divide at an
equal rate, a mosaic of the 45/47 type,
with 2 different genetic patterns distributed
equally throughout the body cells, is produced. If 1 of the cells is nonviable, aneuploidy of a uniform type results as the viable
cell continues to divide. The latter mechanism may account for a considerable proportion of the cases of uniform aneuploidy
reported, although current sentiment favors
meiotic nondisjunction in these cases.
In later mitotic divisions, the number of
variations that may follow nondisjunction
increases almost geometrically. It is quite
probable that nondisjunctive mitoses occur
frequently during human cell division, producing cell lines that either remain phenotypically indistinguishable from neighboring
cells or are completely nonviable. The
potential complexity of mosaicism is reflected in Table 2, in which no less than 13
different sex chromosome mosaics are listed.
Aneuploidy often affects all of the body
cells, including the germ cells. When aneuploid germ cells divide, they in turn produce
some gametes that are aneuploid as a result
of random segregation of the chromosomes
during cell division. For example, the chromosomes in an instance of isolated trisomy
are distributed to 2 classes of gametes, 1 of
which is normally haploid and the other
diploid after reduction division. Fertilization of an abnormally diploid gamete produces an aneuploidy (trisomy) in the zygotes
identical to that of the parent. This is
secondary nondisjunction (Fig. 6).
Translocations
Most of the structural anomalies of chromosomes seem to arise during gametogenesis,
inasmuch as structurally anomalous mosaics
are much rarer than are structural anomalies
that affect all of the body cells.20 Meiotic
crossing-over accounts in part for the frequency of structural anomalies. The role of
the acrocentric chromosomes has been mentioned.
Translocation follows fracture of at least
16
Vol. 89
EGGEN
GERMOn)
PROU
GAMETES
ZYGOTES
F I G . 5 (upper). Nondisjunction in mitosis. T h e potential results of nondisjunction in
the first (A) and second (6) mitotic divisions of a zygote are illustrated. The chromosomes
of normal diploid cells (/) divide to form 2 chromatids united by a centromere (J8). If the
centromere fails to divide, the chromatids are distributed unequally to the daughter cells
(S). In the examples used, the following mosaics result: A, 45 XO/47 X X X ; B, 45 XO/46
XX/47 X X X .
F I G . 6 (lower). Secondary nondisjunction. During the first meiotic division, the chromosomes of a trisomic germ cell are distributed to 2 daughter cells, 1 of which is normally
haploid, the other diploid. The normal daughter cell, during the second meiotic division,
produces normal gametes and normal zygotes are formed (A). The diploid daughter cell
forms diploid gametes and, therefore, triploid (for the affected chromosome) zygotes (B).
2 chromatids and reunion of the fragments
with the "wrong" chromosome. A person
whose cells bear such a chromosome manifests translocation heterozygosis. Translocated
chromosomes undergo mitotic and meiotic
disjunction in the usual manner and can
thus be transmitted from generation to
generation (Fig. 7), as is the case in the
familial transmission of mongolism.36
Translocation carriers may be phenotypically normal and may produce gametes
that, when fertilized, produce zygotes in
which the translocation becomes expressed
pheno typically.
Duplications (Partial Trisomy)
When fragmentation of chromatids occurs,
1 of the fragments may adhere to a chromo-
Jan. 1963
CYTOGENETICS—A
REVIEW
TRANSLOCATION
<fciy
DUPLICATION
F I G . 7 (upper). Translocation heterozygosis. This is a diagrammatic representation of
a 15 (black)/21 (white) translocation. As indicated, the intact Chromosome 21 may be distributed to the gametes in company with either the normal or the translocated Chromosome 15. Possible zygotes from such a translocation a r e : (a) 15, 15/21, 21—phenotypically
normal " c a r r i e r " ; (6) 15, 15/21, 21, 21—mongolism; (c) 15, 15, 21, 21—normal person; (d)
15, 15, 21—monosomy-21, presumably lethal.
The inset illustrates early meiosis in the germ cells of a " c a r r i e r . " The intact Chromosome 21 may accompany either the translocated or the intact Chromosome 15, resulting in the same classes of gametes derived from the original translocation.
F I G . 8 (lower). Duplication ("partial t r i s o m y " ) . The affected locus is illustrated in
white. T h e formation of duplicated and deleted loci in the gametic chromosomes is illustrated. Fertilization produces z}'gotes t h a t are monosomic (above) or trisomic (below)
for the affected loci.
17
18
Vol. 89
EG GEN
some that already carries the same loci that
are present in the fragment. In this event,
in the chromosome pairs that are formed,
1 or several loci are duplicated in 1 chromosome and the same locus or loci are omitted
from the other. Fertilization of a gamete
whose chromosomes bear the duplicated loci
produces a zygote that is functionally trisomic for the affected loci (Fig. 8). Patau 162
has suggested that the term "duplication"
be reserved for the affected gametes and that
"partial trisomy" be applied to the affected
zygotes.
Deletions
Fracture of a chromatid during meiosis
may produce a fragment that fails to reunite
and thus is lost. The resulting incomplete
chromosome is a deleted chromosome.
Isochromosomes
An isochromosome is a chromosome in
which the arms on either side of the centromere are genetically identical. It may be
formed in 2 ways. The first, centric fusion,
follows a simultaneous deletion close to the
centromere in the same arm of each of the
chromosomes in a chromosome pair. The
resulting telocentric forms have terminal
centromeres, are highly unstable, and tend
to unite at their centromere ends.
Anomalous transverse fission of the paired
chromosomes at metaphase, with faulty
reunion of the divided ends, also produces
isochromosomes. Each of these mechanisms
is illustrated in Figure 9.
Inversion
In inversion, a fracture occurs and the
resulting fragment becomes reattached upside down to its point of origin. This phenomenon has often been demonstrated in lower
animal forms, notably in the salivary gland
chromosomes of Drosophila. The large
salivary gland chromosomes are distinctively
banded, and individual chromosome segments are easily recognized. In man, no
such distinctive "labeling" of segments is
present and inversions are, therefore, difficult
to recognize, although there is little doubt
that they occur. This mechanism has been
implicated only once in man,88 to the best of
our knowledge.
Supernumerary Chromosomes
Supernumerary nonfunctional chromosomes and fragments of chromosomes have
been found in some of the lower animal
forms. This phenomenon may be represented
in man104 by some of the rare instances of
asymptomatic trisomy that have been
reported.
AUTOSOMAL ANEUPLOIDY
(Anomalies of the sex chromosomes and
of the autosomes will be discussed separately.)
Isolated Autosomal Monosomy
Monosomy of 1 of the autosomes unaccompanied by another chromosomal abnormality has not been reported in man.
Isolated Autosomal Trisomy
Trisomy of an autosome unaccompanied
by other chromosomal abnormalities is not
infrequent. Three well defined syndromes
have been identified. There are also a number of individual case reports from which
distinct syndromes have not yet emerged.
Mongolism
Lejeune and associates124 described the
first chromosomal anomaly demonstrable
in man when they reported finding trisomy 21 in a number of mongols. Subsequent
investigations have confirmed the association
of this anomaly with mongolism. Nondisjunction during maternal gametogenesis is
the most likely cause in view of the close
relation between advanced maternal age
and mongolism.34 •96 The relation to radiation
has been mentioned. Mongolism associated
with chromosomal mosaicism and inversion
has also been reported.
A familial form of mongolism is also
known, the chromosomal defect in this
instance being a translocation rather than
sporadic nondisjunction. Mongolism is relatively frequent, occurring approximately
once in every 600 births. Its clinical features
are too well known to require amplification
here. There is a close relation between mongolism and acute leukemia,186 the significance of which is discussed with the Ph 1
chromosome below (see " C L O N E S " ) .
Jan. 1968
POSITIVE NEGATIVE
(XX.XXY) 0<y,XO)
XXX OR
XXXY
19
CYTOGENETICS—A REVIEW
XXXX OR
XXXXY
FEMALE
DELETED
X
MALE
ISOCHROMOSOME
X
F I G . 9 (upper)- Isochromosome formation. Centric fusion {left) follows simultaneous
fracture of homologous chromosomes near the centromere (a arrows) and end-to-end
union of the centromere ends of the fragments (6, c). Transverse fission (right) may
produce 2 isochromosomes. The chromosomes in the diagram are homologous chromosomes.
F I G . 10 (lower). Normal and abnormal sex chromatin p a t t e r n s . T h e normal configuration of polymorphonuclear leukocyte nuclear appendages is also illustrated.
Group D Trisomy Syndrome
chromosome involved, the syndrome is
65
There are at least 9 recorded instances ' referred to as "Group D trisomy syndrome."
129, id, 190 0f trisomy involving 1 of the Group The clinical features, recently summarized
D chromosomes (13, 14, and 15). Because by Therman and co-workers,190 include
of the difficulty in identifying the individual defects of the globe (anophthalmia or micro-
20
Vol. 39
EGGEN
phthalmia), cerebral and mental defects,
defects in the falx cerebri, multiple capillary
hemangiomata, hyperextensible thumbs,
cleft palate, hare lip, simian palmar creases,
and occasionally Polydactyly and congenital
heart disease.
Incidence figures are unavailable, but the
syndrome is much less common than mongolism.
Group E Trisomy Syndrome
At least 11 instances of trisomy involving
the Group E chromosomes (16, 17, and 18)
have been described,107 all of them manifesting clinical similarity. The syndrome has
usually been regarded as trisomy of Chromosome 1762 or 18.182 JVIuldal145 has suggested
that all of these may represent trisomy-18,
which has followed unequal crossing-over
and production of some longer-than-normal
(pseudo-17) Chromosomes 18. Without
conclusive evidence incriminating a specific
chromosome, "Group E trisomy syndrome"
is the preferred designation.
The true incidence is unknown, but there
is an apparent correlation between the
anomaly and advanced maternal age. The
clinical features of the syndrome include
variable anomalies of the hands, feet, and
ears (which are usually low set), micrognathia, short webbed neck, duodenal
diverticula, mental retardation, and congenital heart disease of variable severity.
The similarity between the defects of this
syndrome and those of Turner's syndrome
have been commented on by Oikawa and
Blizzard.157
Trisomy of Other A utosomes
Trisomy of other autosomes is infrequent,
and some reported instances are controversial. A presumptive instance of trisomy-3
associated with complete sex reversal has
been reported.167 X / Y translocation, which
would produce a morphologically similar
karyotype, is, however, an equal possibility.
Trisomy of 1 of the Group C autosomes
with mental retardation, mongoloid facies,
and double buccal chromatin has been
reported,171 but, as the authors themselves
state, trisomy-X has not been ruled out. The
fact that there were double buccal chromatin
bodies is strong evidence in favor of the
latter (triplo-X) constitution.
Book and associates25 have described a
family in which both mother and son had
congenital heart disease. The mother was
trisomic for 1 of the Group F (19 to 20)
chromosomes. Her son had similar trisomy,
plus monosomy of 1 of the small acrocentrics.
Although instances of trisomy-2218- 58- 105196
have been described a number of times,
no consistent clinical correlations have
evolved. Trisomy-22 was first reported in
association with the Sturge-Weber syndrome105 but was absent in 14 other instances,93- 95' 104' 123 of the syndrome. Trisomy-22 was found in 2 schizophrenics18196
but was absent in 10 others.18 Trisomy22 has also been reported in association with
congenital myotonia.58 Both supernumerary
chromosome formation104 and partial trisomy162 have been suggested as explanations
of such findings. The possibility that the
extra chromosome might be a Y chromosome
has also been considered.18
Autosomal trisomy may be entirely asymptomatic. Trisomy-19 has been described
in a phenotypically normal male who invariably sired mongols.81 Trisomy-22 may
also represent asymptomatic trisomy.
The relative rarity of autosomal trisomy
and the severity of the associated defects
suggests that autosomal defects of this
magnitude are usually lethal. Chromosomal
defects involving large autosomes (Group
A or B) are extremely rare, as are large
autosomal deletions.
Isolated Autosomal Polysomy
Persons with a chromosome complement
in excess of 47, owing to polysomy of a single
autosome, have not been described.
Complex Autosomal Aneuploidy
Occasional instances of aneuploidy affecting several autosomes or affecting autosomes
and sex chromosomes simultaneously have
been described. These are summarized in
Table 3.
Autosomal Mosaicism
Mosaicism is characterized by the presence of a bimodal, or sometimes a multimodal, chromosome count. Definitive diagnosis
Jan. 1963
21
CYTOGENETICS—A REVIEW
TABLE 3
COMPLEX ANEUPLOIDY
Author
Fraccaro and associates 8 0
Fraccaro and associates 8 0
Book and associates 2 5
Uchida and Bowman 1 0 8
Ford and associates," Hurnden
and associates, 101 Hustinx and
associates, 111 and Lanman and
associates 122
Fraccaro and associates 8 0
Karyotype
21/21/21 and -/\2 (2n = 40)
21/21/21 and -/1G (2n = 40)
Trisomy-F and monosomy-G
(2n = 40)
18/18/18 and X X X (2n = 48)
21/21/21 and X X Y (2n = 48)
8/8/8,11/11/11 and X X V (2n =
49)
Book and Santesson 2 3
Triploidy (3n = 09)
Penrose and Delhanty 1 0 3
D c l h a n t y and associates 5 5
Triploidy (3n = 09)
Triploidy (3n = 09)
of mosaicism depends upon fulfilment of the
following criteria:51 (1) counts differing from
the mode must be too frequent to be attributed to chance; (2) an excess of nonmodal
cells should be found on more than 1 examination and, preferably, in more than 1
tissue; and (3) cells that are nonmodal should
themselves have a similar karyotype.
Autosomal mosaicism is infrequent. We
have been able to find only 2 types reported
and both have been 46/47 mosaics with
autosomal trisomy in approximately half of
the body cells. One type, described by Jacobs
and associates,"4 was encountered in a short
amenorrheic female with normal breasts and
genitalia. The affected cells revealed trisomy-10 or -11. The other type of 46/47
mosaicism has been encountered in mongolism.44' 74' K- Ha- m- m In 1 instance,44 the
mongolism was incomplete. The aneuploid
line revealed trisomy-21 in all.
Structural A nomalies of A utosomes
Most of the reported autosomal structural
anomalies have been translocations. Other
structural defects are much less common.
A utosomal Translocation
Most autosomal translocations have involved the acrocentric chromosomes, pre-
Phenotype
Mongolism
Mongolism
Congenital cardiac disease
I n t e r v e n t r i c u l a r septal
defect,
skeletal anomalies, double chromatin
Mongolism and Klinefelter's syndrome
F l a t occiput, divided scrotum,
cardiac and renal anomalies,
epicanthic folds
Micrognathia, bony s y n d a c t y l y ,
weakness, mental
deficiency,
lipomatosis
Macerated stillborn
Eight-week embryo
dominantly Chromosome 2.1. These translocations have been found in the "carriers"
and in the affected person in familial mongolism; translocations of Chromosome 21
have been to the Group D (13 to 15) and
Group G (21 to 22) chromosomes almost
exclusively. Early reports'3 6 , 78, 81, 104 suggested that the majority of the "carriers"
were females. More recently, however, the
carrier state has been encountered in males97
with increasing freciuency, and a difference
in sex incidence of the carrier state is now
questionable. A more striking finding is that
the production of mongol offspring by male
carriers is a rarity. 64 This suggests that the
genetically normal spermatozoa of male
carriers have a selective ability to fertilize
ova more readily than do spermatozoa that
carry the translocation chromosome.97, m
Other less common translocations involving the autosomes are summarized in
Table 4.
Other Autosomal Morphologic Abnormalities
Deletions, duplications, and isochromosomes, reported with some frequency in the
sex chromosomes, are rare in autosomes.
This fact suggests that structural changes
that are compatible with viability are usually
too small to be readily visible.
22
Vol. 39
EGGEN
TABLE 4
AUTOSOMAL TRANSLOCATIONS
Translocation
Author
Turpin and Lejeune 1 "
Moorehead and associates143
van Wijck and associates206
Carter and associates36 and
others' 8 ' "• 1W
Hamerton and associates97 and
others 36, 64' ,8, 81, 164' ""
Ohno and associates156
German and associates86
Dobson and Ohnuki57
Phenotype
22/13
Mental deficiency
Congenital cardiac anomalies
22/13
Group G/trisomic fragment Neck webbing, ear malformations,
of 17 or 18
skeletal defects, mild epilepsy
Familial mongolism
21/21
21/15
Familial mongolism
21/13
?2/long arm of 2
Group A/Group C
Marfan's syndrome
Waldenstrom's macroglobulinemia
Convulsive disorder. (Defect suspected on the basis of atypical
chromosome constrictions.)
Recently, Patau and associates162 have
suggested the term "partial trisomy" for
minute duplications that they believe may be
responsible for many congenital defects not
associated with obvious aneuploidy. Duplication of 1 or several loci, producing functional trisomy of these loci has been implicated in the Sturge-Weber syndrome162 and
in some instances of congenital disease that
resemble Group E trisomy syndrome but
lack obvious aneuploidy.181, 206
Presumptive isochromosome formation
has been encountered in association with
Waldenstrom's macroglobulinemia by several investigators. 17, 27, S6, 158 It has been
suggested86, 158 that the very large extra
autosome encountered may represent an
isochromosome of the long arm of Chromosome 2. We have not found any other
reported instances of formation of autosomal
isochromosomes.
Ring autosomes have been encountered in
peripheral blood following x-irradiation,193
but these have not, apparently, been
self-perpetuating. A recent report127 of a
ring chromosome X that was apparently
self-perpetuating is referred to in Table 6.
A presumed instance of pericentric inversion of Chromosome 21 in mongolism
has been cited.88
Clones
A clone has been defined as an asexually
produced population of which all members
have been derived from 1 and the same
progenitor exclusively by nonsexual reproduction.186 Mosaics fall within the scope of
this definition, inasmuch as they arise during
nondisjunctional mitosis (asexual). The term
may, however, be used to designate a genetically atypical cell population arising within
a differentiated body tissue and is so applied
by many workers with reference to tumors.
In our classification, we are using the term
in this more restrictive sense, to serve as a
convenient method of distinguishing singletissue abnormalities from those of the body
cells as a whole.
There is a distinction to be made between
clones and chimeras. The latter are organisms
that have genetically different tissues in
close apposition as a result of grafting. Human blood group chimeras have been described in twins following transplacental
transmission (grafting) of blood cells.26, Mi5o, 2u All have been A/O cell mixtures and
have been detected by differential blood
typing. In 1 instance,211 the cells were cultured and were found to have differing sex
chromosomal patterns.
Many human malignancies have the
characteristics of clones; their aneuploidy is
usually random and unpredictable, a characteristic that has been used112 as a diagnostic
aid in the study of effusions. An exception to
the generally random aneuploidy of neoplasms is the Ph 1 chromosome found in
chronic myelocytic leukemia by Nowell and
Jan. 1968
CYTOGENETICS—A
Hungerford153 and by Baikie and associates.9
The abnormal chromosome is found in
blood and bone marrow cultures from
affected patients but not in other body tissues. The Ph 1 chromosome may be either a
deletion of the long arm of 1 of the small
acrocentrics1' 1M or a translocation of 1 of
the small acrocentrics to Chromosome 14 or
15.169
The Ph 1 chromosome is absent in acute
myelogenous leukemia116 and in the terminal
acute phase of chronic myelocytic leukemia.
Patients with chronic myelocytic leukemia
who have had unusually prolonged and
benign courses (and usually irradiation
therapy) also lack the Ph 1 chromosome.
An increased incidence of acute leukemia
in mongolism (trisomy-21) is well established. Recently, a number of authors 8, 120,
133
have reported an association between a
variety of chromosomal defects and blood
dyscrasias. Many of these have been sex
chromosomal defects. There is also an increasing number of reports101' l u ' 122' 137'
114
linking sex chromosomal defects and
trisomy-21. These recent findings open
interesting speculative possibilities, although
the relations are still too infrequent to be of
statistical significance. One distinct possibility is the existence of a leukopoietic locus
on Chromosome 21, duplication of which
(mongolism) produces acute leukemia, and
deletion of which (Ph1 chromosome) produces
chronic leukemia.
Investigations of acute leukemia10' 116' 121
have not always revealed consistent structural changes. It is possible that each morphologic variety of leukemia has several
etiologic subtypes;121 the resultant multiplicity of possible etiologies would complicate investigations of chromosomal patterns.
SEX
CHROMATIN
The nuclear chromatin test is an important screening test for detecting sex chromosomal abnormalities14 and is most easily
performed on buccal preparations. Cells
from urinary sediment, amniotic fluid, or
vaginal smears may also be used. The nuclear sex chromatin is a distinctive mass
closely applied to the inner aspect of the
nuclear membrane; it measures approxi-
REVIEW
23
mately 1.2 by 0.7 /*."• 12 In smears from a
normal woman or girl, 40 to 60 per cent of
the nuclei will have a chromatin body. In
skin biopsies, chromatin bodies are somewhat more numerous (60 to 80 per cent of
the epithelial cells). Minute chromatin
bodies have been found in men and boys by
some166, l74 ' 176 but not all investigators.
The derivation of the nuclear sex chromatin has been the subject of considerable
speculation. Barr's original work led him to
believe that the chromatin mass was derived
in part from each of the X chromosomes.
Many subsequent theories have been advanced to explain the origin of the chromatin
body, and these have been critically reviewed by Miles.134
The number of chromatin masses in each
nucleus is in the order of n — 1 where n
is the number of X chromosomes present.
Reports at apparent variance with this
general relation167 have often included associated anomalies that might mask the presence of a second X chromosome. Alterations
of the n — / relation seen in triploid XXY
persons (69 chromosomes) may be the result
of altered ratios of autosomes to sex chromosomes.24' "• 139
Such a numerical relation seems most
consistent with the concept that only 1 of
the X chromosomes in any cell is "active" 154
and that the "inactive" X or X's become
applied inertly to the nuclear membrane.
This concept is supported by the findings of
Ohno and Makino,165 who have found that
the germ cells of female cats, which have
euchromatic X chromosomes, have no nuclear sex chromatin. Somatic cells from the
same cats, which have heterochromatic X
chromosomes, do have nuclear sex chromatin.
Regardless of its origin, the nuclear sex
chromatin seems to be related to the number
of X chromosomes present rather than to
the genetic sex of the person (Fig. 10). For
this reason, the noncommittal designations,
"chromatin-positive" and "chromatin-negative," should be used in preference to "male"
and "female" in reports of these tests. Inasmuch as males may be chromatin-positive
(Klinefelter's syndrome) and females chromatin-negative (Turner's syndrome), infer-
24
ence of true genetic sex from the results of a
chromatin test may be incorrect.
Consistent variations in the size of the
chromatin body may be significant. Care
must be taken in reporting such variations,
however, inasmuch as artifactual variations
in size have been known to follow antibiotic
therapy.184
The nuclear appendages ("drumsticks")
of polymorphonuclear leukocytes are found
in genetic females in 3 per cent or more of
the polymorphonuclear leukocytes. The
appendages are racquet- or club-shaped and
have distinctly bulbous ends. Smaller,
cylindrical appendages are occasionally
noted on the nuclei of the polymorphonuclear leukocytes of males, but these are less
prominent and do not have bulbous ends.
The origin of these appendages is obscure.
They may represent secondary sex characteristics rather than direct chromosomal
derivatives.
ABNORMALITIES
Vol. 89
EGGEN
OF T H E S E X
CHROMOSOMES
Reports of sex chromosomal abnormalities
are more numerous and include a greater
variety of defects than those of autosomal
abnormalities. There are probably several
reasons for their preponderance. Most
significant is the apparent lethal nature of
autosomal defects of large magnitude. It
seems that the sex chromosomes subserve
less vital functions than do the autosomes.
Many of the high magnitude anomalies
found in the sex chromosomes (e.g., polysomy) have not yet been encountered in the
autosomes.
To some degree, the difference in prevalence may be more apparent than real. The
sex chromatin test provides a convenient
method of screening likely candidates for
karyotyping from large numbers of persons.
No such convenient screening test is available for the detection of autosomal defects.
Genetic sex in man is determined by the
Y chromosome, and persons who possess a
Y chromosome are regarded as genetic males
regardless of the number of X chromosomes
that may be present. The functions of the
Y chromosome are not, however, entirely
clear. It does not seem essential to the development of testicular tissue,177 inasmuch
as true hermaphrodites (who have identifiable, if imperfect, testicular tissue) rarely
have a Y chromosome,109' 135,2M the majority
possessing only X chromosomes.4, 73, 100' "°
Secondary male sex characteristics (e.g.,
adrenal virilism) are also not under the sole
control of the Y chromosome. Although a
virtually perfect female phenotype may be
encountered in genetic males (testicular
feminization), a near perfect simulation of
male phenotype (i.e., scrotal testes and
penile urethra with no perineal defects) has
not been encountered in the absence of a Y
chromosome. Rare reports that seemingly
contradict this view21' 94 have listed associated chromosomal defects that could
readily mask a Y / - translocation, as has
been pointed out by others.87 The complex
functional interrelation of the X and Y
chromosomes has been explored by several
investigators.14C' 154
Recent findings in respect to the anomalies
of the sex chromosomes suggest useful
changes in nomenclature. Anomalies that
have a well established chromosomal basis
should be designated accordingly. Studies of
Turner's syndrome particularly reveal that
several distinct genetic entities should
be differentiated. The apellation "male
Turner's syndrome," 0 although admittedly
descriptive, should be modified in the light
of recent findings, such as the similarities between Turner's syndrome and the Group E
trisomy syndrome.157
Many of the sex chromosomal anomalies
are common in mentally defective populations. 16 ' 68' »'• ,19' 131' ,44 Mental deficiency of
sufficient degree to require institutional
treatment seems to correlate well with the
number of X chromosomes present,81, 131
although this relation is not entirely unchallenged.91' 98 A correlation between sex
chromosome defects and blood dyscrasias is
increasingly noted, although its statistical
significance is still dubious. 8, 101, 120, 133
Sex Chromosomal Monosomy
Isolated monosomy of the sex chromosomes has been recognized only in the XO
constitution, YO presumably being lethal.
Patients of the XO type have chromatinnegative Turner's syndrome, the genetic
Jan. 1968
CYTOGENETICS—A
pattern of which was anticipated as early as
1954 by Polani and co-workers.166
Whether the XO pattern is derived by
mciotic (gametic) or mitotic nondisjunction
is uncertain. It is significant that a correlation between maternal age and incidence is
absent in Turner's syndrome. The sexlinked, blood group factor Xg132 that was
recently discovered should provide a valuable reference system for determining the
origin of the defect. Another significant recent discovery is that of a ring chromosome,
presumably a ring X, in an instance of
gonadal dysgenesis.129 Such ring forms are
highly unstable, and development of anomalous configurations of this type may result
in chromosome loss early in zygotic development.
Eighty-five per cent of the patients regarded as having Turner's syndrome are
chromatin-negative. The remainder, who do
not fit the classical description of Turner's
syndrome, are chromatin-positive and manifest mosaicism or other abnormalities.
Chromatin-negative females are encountered
in approximately 0.3 of every 1000 live
births.180' 140' 144 '
The typical patient with Turner's syndrome01' ir'7 is a phenotypic female of short
stature who exhibits sexual infantilism,
neck webbing, and dysgenesis of the gonads,
which are represented by streaks of ovarian
stroma. These patients are amenorrheic and
sterile (although fertility with the XO constitution has been reported). 7 Bony and
soft-tissue anomalies are also present and
include shield-like chest, cubitus valgus,
micrognathia, high-arched palate, low set
ears, hypertension, peripheral lymphedema,
and aortic coarctation. Many of these patients have subnormal intelligence, which
is not always, however, of a degree of severity to require institutional care. Of mentally
subnormal persons, 0.4 per cent have an XO
pattern. 40
Many of the patients designated as "chromatin-positive Turner's syndrome" have
had chromosomal mosaicism of the 4 5 X 0 /
4(:>XX type. In addition to being chromatinpositive, these patients lack important
stigmata that characterize the classical
Turner syndrome. De Grouchy and co-
25
REVIEW
workers89 have reviewed a number of such
cases and have noted that, although the
patients are short and amenorrheic, they do
not manifest any of the bony and soft-tissue
defects that are equally characteristic of the
true Turner's syndrome.
Another moderately well defined intermediate form of gonadal dysgenesis accompanying XY/XO mosaicism has been reported19' 109' 114' 136 and has been summarized
recently by Willemse and associates.'209
Affected patients have been chromatinnegative and have manifested gonadal
dysgenesis with varying degrees of masculinization. To categorize such forms of gonadal
dysgenesis as variants of Turner's syndrome
serves no useful purpose and tends to obscure
the highly characteristic features of classical
XO Turner's syndrome. Furthermore, such
a practice minimizes the striking differences
in chromosomal pattern that are encountered
in the various types of gonadal dysgenesis.
We have, therefore, adopted the practice
of referring to such persons as "chromatinpositive" or "chromatin-negative gonadal
dysgenesis" and reserving the term
"Turner's syndrome" for the classical XO
form.
Isolated Sex Chromosomal Trisomy
The presence of 3 sex chromosomes,
whether X or Y, constitutes sex chromosomal trisomy.
Trisomy-X
(Triplo-X)
The X X X pattern was originally reported
in man as an example of the "superfemale"
state," 3 a term borrowed from work on
Drosophila. This term is a misnomer and
X X X patients are better referred to as
"trisomy-X" or "triplo-X." The patients
may be amenorrheic and sterile, and varying
degrees of mental deficiency are often present.83' 131 Such patients comprise 0.7 per cent
of institutionalized mental defectives.72
Occasional instances of trisomy-X in which
no phenotypic changes were present have
been detected when double chromatin bodies were found on routine buccal smears.200
A recent analysis of 18 reported instances
of trisomy-X" 7 has emphasized the lack of a
consistent clinical appearance associated
2G
Vol. 39
EGGEN
with this chromosomal constitution. The
condition is not exceptionally common
(0.8130 to 1.3144 per 1000 live births).
XX Y (Klinefelter's Syndrome)
Chromatin-positive Klinef elter's syndrome
is the most common anomaly of the sex
chromosomes, occurring in 3 of every 1000
births' 30, H0 and accounting for approximately 1 per cent of institutionalized mentally defective men and boys.72 Approximately 3 per cent of the men in the average
sterility clinic have Klinefelter's syndrome.
Although many instances of XXY are
thought to arise from gametic nondisjunction, disjunction in an early zygotic division
may be at fault. Use of the sex-linked blood
group as a reference system should be
equally valuable in assessing this problem.
Characteristic of Klinefelter's syndrome
is primary microorchism, with small, firm
scrotal testes, manifesting tubular dysgenesis
histologically. The patients are usually
sterile and have positive buccal chromatin
and leukocyte patterns. Eunuchoidism and
gynecomastia are frequent, but not invariable findings. Subnormal intelligence is
noted in approximately 25 per cent of these
patients." 9
XYY
There are at least 3 instances18 • 148, 173 in
which this form of trisomy has been considered; however, in only 1 of these173 was
XYY regarded as the most probable chromosomal pattern. The patient was described
as an apparently normal male who sired 8
children, of whom 2 were abnormal (mongolism, amenorrhea).
Isolated Sex Chromosomal Polysomy
Higher degrees of polysomy have been
known to affect the sex chromosomes; they
include instances of XXXX, 32 XXXY, 72
XXXXY, 82 and X X Y Y " The higher polysomes with a Y chromosome have had
Klinefelter's syndrome with a degree of
mental deficiency roughly proportional to
the number of X chromosomes.72 A survey
of reports of the X X X X Y form82 indicates
that this form may be sufficiently distinctive
to merit consideration as a separate syn-
drome that embraces mental and skeletal
defects and complete tubular dysgenesis.
Combined Autosomal and Sex Chromosomal
Anewploidy
Most instances have been variants of
Klinefelter's syndrome and have been summarized in Table 3.
Sex Chromosome Mosaics
The review of X O / X X mosaicism89 has
been cited. Other mosaics, many of which
are examples of gonadal dysgenesis, are
summarized in Table 5.
Structural Alterations of Sex Chromosomes
Most of the structural anomalies that can
be recognized in experimental animals have
been detected in the sex chromosomes.
Apparent discrepancies between nuclear sex
chromatin patterns and the number of X
chromosomes found may, in some instances,
be the result of unrecognized structural
alterations. Structural anomalies have variable phenotypic sequelae, which are summarized in Table 6.
Sex Abnormalities with no Chromosomal
Basis
Many intersex states are not due to detectable errors in chromosome pattern.
Some, such as adrenal virilism and testicular
feminization, seem to have a hormonal basis.
Chromatin and chromosome studies are
extremely valuable in assessing genital
ambiguity in infants, where the distinction
is of great practical importance. The study
of intersex has many complexities, some of
which are the consequence of variations in
terminology. Intersex is best regarded as a
difference between the apparent sex of a
person and his chromosomal (not chromatin)
sex. Recent articles 3,21 ° illustrate some of the
changing concepts of intersex.
CLINICAL
APPLICATIONS
Cytogenetic technic and knowledge are
sufficiently advanced to be of proved value
in several clinical situations. In assessing
sterility, the buccal chromatin test and
karyotyping can be most illuminating. A
study by Jacobs and associates114 has dem-
Jan. 1963
27
CYTOGENETICS—A REVIEW
TABLE 5
S E X CHROMOSOME M O S A I C S
Author
Chromatin
Mosaic Pattern
Phenotype
De Grouchy and associates 8 9
and others 3 9 ' 114
Willemse and associates 2 0 9
and others 1 9 ' 114, 136
45 XO/46 X X
Positive
Gonadal dysgenesis
45 XO/46 X Y
Negative
Hii'schhoi-n and associates 1 0 9
and others 1 3 5
Jacobs and 'associates 114, U 5
45 XO/46 X Y
Negative
I n t e r m e d i a t e of
gonadal
dysgenesis and testicular
feminization
True hermaphrodite
45 XO/47 X X X
Positive
Cooper and associates 5 0 and
others 114
Waxman and associates 2 0 3
45 XO/47 X Y Y
Negative
Gonadal dysplasia, low intelligence
Amenorrhea
Jacobs and associates 1 1 5
Ferguson-Smith and associates 7 3
De Grouchy and associates 9 0
46 XX/47 X X Y
46 X X / 4 7 X X X
Positive
Positive
True hermaphrodite (ovotestis seen)
Ivlinefelter's syndrome
T r u e hermaphrodite
46 X X / 4 7 X X X
Positive
Baikie and associates 8 and
others 1 0 6 , 131
Maclean and associates 1 3 1
45 XY/47 X X Y
Positive
48 X X X Y / 4 9 X X X X Y
Positive
Jacobs and associates 114
Hayward and Cameron 1 0 6
45 XO/46 X X / 4 7 X X X
45 XO/46 X X / 4 7 X X X
Positive
Positive
Jacobs and associates 114
Maclean and associates 1 3 1
Maclean and associates 1 3 1
45 XO/46 XY/47 X Y Y
45 XO/46 XY/47 X X Y
46 XY/47 X X Y / 4 8 X X Y Y
Negative
Positive
?46 X X / 4 6 X Y
onstrated that 25 per cent of patients with
primary amenorrhea will be found to have a
chromosomal defect. Discovery of such a
chromosomal abnormality early in the
investigation can save the patient the time
and expense of repeated courses of hormone
therapy and prolonged laboratory study.
Successful correction of the intersex states
depends on the early and accurate diagnosis
of genetic sex, which is best accomplished by
means of cytogenetic methods. Accurate
diagnosis and appropriate therapy often
require karyotyping and should not be based
on buccal smears alone, although the latter
are a very useful screening procedure.
Rendering a prognosis to the unfortunate
parents of a mongol or other congenitally
deformed infant is facilitated bj r cytogenetic
studies.34, 84, 96 Studies of both parents and
of the affected infant differentiate sporadic
Severe oligomenorrhea
(?Stein-Levinthal)
Klinefelter's syndrome
Mental
deficiency—Klinefelter's syndrome
Gonadal dysgenesis
Gonadal dysgenesis—
Hirschprung's disease
Amenorrhea
Mental deficiency
Mental deficiency
and familial defects in many instances and
help to make prognosis reliable.
Future developments in cytogenetics76 will
depend upon refinements in technical
methods. Until individual chromosomes and
different chromosome segments can be
identified with a high degree of certainty,
genetic mapping in man will be delayed.
Mapping technics that can be applied to
lower animals and that may be of value in
man have been reviewed by Sachs and
Krim.170
At present the sex-linked loci are among
the few that have been assigned to a specific
chromosome. The X chromosome is known,
or presumed, to bear loci governing a blood
group (Xg132), hemophilia, red-green color
blindness, glucose-6-phosphate dehydrogenase,195 stature (in part), sex chromatin, and
gonadal cortical development. Attempts
28
Vol. 89
EGGEN
TABLE 6
STRUCTURAL A B N O R M A L I T I E S OF S E X CHROMOSOMES
Author
Phenotype
Abnormality
Edwards60
X / X translocation
Oikiiwa and Blizzard 1 5 7
Turpin and Lejeune 1 9 7
De Grouchy and associates 9 0 and
others 1 1 5
Jacobs and associates 1 1 4
? X / Y translocation
?21/Y translocation
Deletion long arm of X
Muldal and Ockey 146 '
Deletion long arm o Y
149
Deletion short arm of X
Conen and associates 4 7
Deletion Y
Jacobs and associates 1 1 4
Blank and associates 2 0
others 7 9 ' '">
Lindsten and Tillinger 127
PDuplication X
Isochromosome X
and
Engel and Forbes 6 6
Blank and associates 2 0 and Lindsten 1 2 6
De Grouchy and associates 9 0
Waxman and associates 2 0 3
Vaharu and associates 2 0 0
Ring X
Amenorrhea, normal intelligence,
atypical nuclear chromatin (possible X / 9 )
Complete sex reversal
Gonadal dysgenesis, small chromatin bodies
Amenorrhea,
small
chromatin
bodies
Male, hypospadias, and muscular
dystrophy
Female phenotype, testicular tissue
Amenorrheic female
Chromatin-positive gonadal dysgenesis
Chromatin-negative oligomenorrhea, cubitus valgus
Short, amenorrheic, obese female
Isochromosome del 3 ted long
arms X
45 XO/46-isochromosome X mo- Chromatin-positive gonadal dysgenesis
saic
Oligomenorrheic female, follicular
4G X X / 4 6 X-deleted x mosaic
atresia
T r u e hermaphrodite
(?XX/XY
?46XX/4GX-deleted x mosaic
mosaic)
46XX/47XX plus fragmen t (mo- Female—gonadal dysplasia, enlarged phallus
saic)
have been made to localize these loci on the
X chromosome.187 The many complexities of
chromosome-mapping that must be considered in such attempts have been reported
by several authors. 138 ' l54, 165, 195
SUMMARY
The increasing importance of cytogenetics
to clinical pathologists and clinicians is
indicated.
Normal mechanisms of mitosis and meiosis
are reviewed in some detail, and some of the
errors to which they are subject are described. The most common error is nondisjunction. Other abnormal mechanisms
discussed are deletion, duplication, isochromosome formation, inversion, and the alteration of genetic pattern by viral infection.
Well established trisomy syndromes,
including mongolism, Group D trisomy
syndrome, and the Group E trisomy syndrome, are presented in detail.
The origin and significance of the Burr
chromatin body is discussed, and its relation
to the X chromosome is presented.
Abnormalities of the sex chromosomes are
reviewed, and the differences between classical Turner's syndrome and some of its
variants are discussed. Klinefelter's syndrome is discussed.
Within the near future, cytogenetic technics may be expected to become commonplace in the clinical laboratory.
APPENDIX
TECHNICAL METHODS
DEMONSTRATION OF NUCLEAR SEX
CHROMATIN
Smears may be prepared from urinary
sediment,37 amniotic fluid, or vaginal31 or
Jan. 1963
CYTOGENETICS—A
buccal"2 scrapings. Buccal smears are the
most satisfactory, inasmuch as there is little
autolysis (as in amniotic and urinary preparations) or nuclear pyknosis (as in some
vaginal preparations). We have obtained
acceptable preparations by means of the
Papanicolaou technic, processing buccal
smears with routine vaginal smears. The
method recommended by Barr" yields preparations of great clarity, as does the use of
aceto-orcein.175, 1%
Collection of Specimens
Scrapings are taken from the inner aspect
of the cheek by means of a tongue blade or a
metal spatula under firm pressure (but not
sufficient to cause bleeding or oozing). Cells
are spread uniformly on a glass slide, which
is immediately immersed in fixative (equal
parts of 95 per cent ethyl alcohol and ether).
Gently pressing the coverslip on the smears
before fixation flattens the nuclei and makes
the chromatin mass easier to see.
Staining Method {Ban ")
Reagents:
Stock thionin:
Thionin
Ethyl alcohol, 50 per cent
Stock buffer:
Sodium acetate
Sodium b a r b i t u r a t e
Distilled water to
Working s t a i n :
0.1 N HC1
Stock buffer
Stock thionin
1.0 Gm.
100.0 ml.
9.714 Gm.
14.714 Gm.
500.0 ml.
2.
3.
4.
5.
0.
7. Pass slides through 2 changes of distilled water of 5 min. each.
8. Stain slides with the working solution
of thionin for 5 min.
9. Pass slides through 3 increasing concentrations of ethyl alcohol (70 per
cent, 95 per cent, and absolute) for
1 min. each.
10. Clear in xylol and mount.
Reporting
In the normal woman or girl, 40 to 60 per
cent of the cells will have a characteristic
chromatin body that is absent from the cells
of normal men and boys. Report the preparations as "chromatin-positive" or "chromatin-negative," and describe any abnormalities of size or number.
EXAMINATION
OF
NEUTROPHILS
Three per cent or more of the neutrophils
in women and girls bear characteristic accessory lobes on their nuclei. These lobes
are absent from the neutrophil nuclei of
normal men and boys. Any routine staining
method, such as Wright's or Giemsa's, is
satisfactory. A sufficient number of cells
should be examined to allow at least 6 such
accessory lobes ("drumsticks") to be demonstrated. If none are present in 500 cells, a
report of "male pattern" is made.
P E R I P H E R A L BLOOD
32.0 ml.
28.0 ml.
40.0 ml.
This working stain will last for 6 to 8 weeks
without loss of staining quality.
\.
29
REVIEW
Method
Immerse slides in absolute ethyl
alcohol for 3 min.
Immerse slides in 0.2 per cent perlodion solution for 2 min. (to enhance
adherence of cells in later steps).
Dry slides in air for 15 sec.
Immerse slides in 70 per cent ethyl
alcohol for 5 min.
Pass slides through 2 changes of distilled water of 5 min. each.
Hydrolyze with 0.1 N HC1 at 56 C.
for not more than 5 min.
CULTURE
This method is a modification of the
technics of Ferguson-Smith and Johnston,71
Hungerford and co-workers,"0 and Edwards
and Young.r'3 More rapid direct methods
have been described,121 but we have not had
any experience with them.
The reagent used is aceto-orcein stain.
Using a reflux condenser, dissolve 2.0 Gm.
of natural orcein in 45 ml. of glacial acetic
acid. Add 55 ml. of water while the solution
is still warm. The stain must be filtered
before it is used.
Method
Sterile conditions must be maintained
throughout the culture steps of the procedure.
]. Collect from 5 to 20 ml. of blood in a
heparinized syringe. Smaller speci-
30
Vol. 89
EGGEN
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
mens (e.g., from heel pricks) should
be increased to 5 ml. by the addition
of AB serum. A useful container that
facilitates separation of cells has been
devised by Kaijser." 8
Add 0.5 ml. of phytohemagglutinin,
and allow the blood to stand for 2 hr.
(not longer) or centrifuge it gently at
200 r.p.m.
Add 1 to 2 ml. of the supernatant
fluid to medium (Difco 199) to which
penicillin (250 n per ml.) and streptomycin (100 Mg- per ml.) have been
added. A final cell concentration of 1
to 3 million cells per ml. should be
obtained.
Incubate at 37 C. for 3 or 4 days.
Add colchicine to the culture medium
to produce a final concentration of
0.0001 per cent, and incubate for
another 2 hr.
Transfer the culture material to a
centrifuge tube, and centrifuge it
gently (400 r.p.m.) for 5 min.
Pipet off the supernatant, add 2 to
3 ml. of 1 per cent sodium citrate, and
resuspend the cells for 3 min.
Centrifuge gently (200 r.p.m.) for 4
to 5 min. and pipet the supernatant.
Fix in acetic alcohol (3 parts absolute
ethyl alcohol and 1 part glacial acetic
acid), adding the reagent slowly,
drop by drop, with gentle agitation.
Allow to stand for 10 min. and centrifuge gently.
Pipet- off the fixative and place the
cells in 45 per cent acetic acid in the
refrigerator for at least 1 hr. (preferably overnight).
Pipet the supernatant and again treat
with acetic alcohol fixative.
Place a few drops of the cell suspension
on several coverslips and allow to dry.
Stain.
Staining Method
The method described yields permanent
slides. Stains for temporary mounts that are
satisfactory for most routine studies will be
outlined later.
1. Stain with aceto-orcein for 25 min.
2. Pass slides through 2 changes of tertiary butyl alcohol of 1 min. each.
3. Pass slides through 1 more change of
tertiary butyl alcohol of 2 min.
4. Immerse slides in 1:1 tertiary butyl
alcohol to xylene solution for 2 min.
5. Immerse slides in xylene for G min.
6. Immerse slides in xylene for 2 additional min., and mount them while
they are still wet, using balsam or
Permount.
B O N E MARROW
CULTURE
This method is a modification of that
suggested by Patau. 160
1. Add 0.25 ml. of marrow to approximately 4.0 ml. of medium (Hanks'
saline solution and fasting, fat-free
AB serum in equal parts, with penicillin and streptomycin added).
2. Allow to stand at room temperature
for 24 hr.
3. Incubate for 5 hr. at 37 C.
4. Add colchicine in a final concentration
of 0.0001 per cent, and incubate for
another 2 hr. at 37 C.
5. Centrifuge gently and aspirate the
supernatant.
6. Add 2 or 3 ml. of 1 per cent sodium
citrate and allow to stand for 3 min.
7. Centrifuge gently; aspirate the supernatant, fix the cells with 1:3 acetic
alcohol, and allow to stand for 10 min.
8. Centrifuge gently; aspirate the supernatant, add 45 per cent acetic acid,
and refrigerate overnight.
9. Aspirate the supernatant and add
fixative (acetic alcohol).
10. Place small drops of the cell suspension on coverslips, allow to dry, and
invert the coverslips over a drop of
aceto-orcein stain on a glass slide.
Seal the edges of the preparation with
appropriate cement.
This method yields temporary preparations of good technical quality. Some authors
have eliminated the colchicine,108 and some
have even used marrow directly, without
culturing.171 Schiffer and associates170 use
acridine orange staining and fluorescence
microscopy, believing that fewer trouble-
Jan. 1.963
CYTOGENETICS—A
some artifacts are encountered with this
method.
C U L T U R E OF BIOPSY MATERIAL FROM
SKIN
Of tissue that is suitable for culture, skin
is the most accessible. The tissue culture
method outlined serves well for fascia lata
and for fetal material. The method of
Hirschhorn and Cooper108 is a good one.
Collection of Specimens
Skin tissue for biopsy is obtained under
sterile conditions and with the use of local
anesthesia. The area should be anesthetized
by deep injection rather than by the intradermal technic. The fragment obtained
should measure approximately 5 by 4 by 2
mm. This fragment is placed in sterile saline
solution until itisused (refrigerate itif culture
is not anticipated within a few hours).
Culture Method
The specimen for biopsy is divided, under
sterile conditions, into fragments having
maximal diameters of 0.5 to 1.0 mm. The
fragments are placed on or near the coverslips, and medium is added until it nearly,
but not quite, covers the explants. The
medium consists of Difco medium 199 (70
per cent), single donor AB serum (20 per
cent), and chicken embryo extract (10 per
cent) with antibiotics added.
Incubate at 37 C. in a 5 per cent C0 2
atmosphere, and change the medium (under
sterile conditions) 3 times a week without
disturbing the cells.
In 1 to 3 weeks, when growth appears
active, coverslip preparations may be made,
using the technic outlined below. Relatively
few mitoses can be expected.
1. Remove the remainder of the cells
from the dishes and treat them with
a 1:1 solution of Versene and trypsin
in Hanks' solution (without calcium or
magnesium).
2. Centrifuge the cell suspension gently
for 5 min.
3. Aspirate the supernatant fluid and add
2 to 10 ml. of medium, depending on
the cell density.
4. Distribute aliquots of the material into
31
REVIEW
sterile Petri dishes containing sterile
coverslips.
5. Incubate these preparations for 3 or 4
days at 37 C. in a 5 per cent C0 2 atmosphere.
6. Add colchicine to a final concentration
of 0.0001 per cent and incubate for
another 3 or 4 hr.
7. Remove the coverslips and treat according to the staining method given
below.
This method is also highly satisfactory for
the culture of bone marrow.
Staining Method
1. Immerse coverslips in 0.7 per cent
sodium citrate for 10 min.
2. Immerse coverslips in 2 per cent acetoorcein stain for 5 min.
3. Invert coverslip over a glass slide, and
squash the cells gently.
4. Examine the preparation microscopically in order to determine if squashing is sufficient.
5. Seal the coverslip to the slide with an
appropriate cement.
This method produces preparations that
are temporary, but entirely satisfactory
for routine examination.
KARYOTYPING
Regardless of the source of the material,
or the culture and staining methods used,
final karyotyping is similar. The slides are
examined under oil immersion, and chromosome counts are made on as many adequate
metaphase figures as can be found. Photographs of good metaphase figures are made
and enlarged, and the chromosomes are cut
out of the photographs and arranged in
order of descending length and in homologous pairs.
SUMMAIirO I N
INTERLINGUA
Es signalate le crescente importantia del
cytogenetica pro le pathologo clinic e pro le
clinico practicante.
Le mechanismos normal del mitose e del
meiose es revistate in considerabile detalio.
Certes del errores que occurre in illos es
describite. Le error le plus commun es nondisjunction. Altere mechanismos anormal
32
Vol. 39
EOGBN
que es includite in le discussion es deletion,
duplication, formation de isochromosomas,
inversion, e le alteration del configuration
genetic per infection virusal.
Es presentate in detalio plure ben-establite
syndromes de trisomia. Istos include mongolismo, syndrome de trisomia de Gruppo D,
e syndrome de trisomia de Gruppo E.
Le origine e le signification del corpore
chromatinic Barr es discutite, e su relation
con le chromosoma X es analysate.
Anormalitates del chromosomas de sexo es
revistate. Le diiTerentias inter le classic
syndrome de Turner e certes de su variantes
es discutite. Etiam le syndrome de Klinefelter es discutite.
U es a previder que in le proxime futuro
technicas cytogenetic va esser parte del
routine del laboratorio clinic.
Boston: Little, Brown and Co., 1960, p p .
334-336.
12. BARR, M . L . : Sexual dimorphism in interphase nuclei. Am. J . H u m a n Genet., 12:
118-127, 1960.
13. B A R R , M . L., AND BERTRAM, E . G . : A mor-
phologic distinction between t h e neurones
of the male and female, and t h e behavior
of the nucleolar satellite during accelerated
nucleoprotein synthesis. N a t u r e , London,
163: 676, 1949.
14. B A R R , M . L., AND C A R R , D . H . : Sex c h r o m a -
tin, sex chromosomes and sex anomalies.
Canad. M.A.J., 83: 979-986, 1960.
15. B A R R , M . L., SHAVER, E . L., C A R R , D . H . ,
AND P L U N K E T T ,
16. B E N D E R , M . A., AND GOOCH, P . C : An un-
usually long human Y chromosome.
cet, 2 : 463-464, 1961.
17. B E N I R S C H K E ,
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F . W . : A new chromosome abnormality
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2. A N D R E S , V. G., P R A D E R , A., H A U S C H T E K ,
E . , SciIARER, K . , SlEBENMANN, R . E . ,
AND H E L L E R , R . : Multiple sex chromatin
and complex chromosomal mosaic in a boy
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Helvet. paediat. acta, 15: 515-532, 1960.
3. A S H L E Y , D . J . B . , AND J O N E S , C. H . : Sex
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E.,
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87.
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93. GUSTAVSON,
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The
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95. H A L L , B . : T h e chromosomal constitution of
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77. F O R D , C. E . , J O N E S , K . W., M I L L E R , O. J . ,
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Vol. 89
EGGEN
AND C A R T E R ,
C.
O.:
Chromosome
studies in detection of patients with high
risk of second child with Down's syndrome.
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F . , BRIGGS, S. M . , AND P O L A N I , P . E . :
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