2
Methods of Studying Human
Chromosomes and Nomenclature.
The Normal Human Karyotype
GOPALRAO V.N. VELAGALETI AND VIJAY S. TONK
Ever since the elucidation of the correct human chromosome number (2n ⫽ 46)
by Tijo and Levan [1], clinical cytogenetics has become an important branch of
medical genetics. It was natural that this epoch-making discovery was soon fol-
lowed by discovery of various numerical chromosomal abnormalities such as tri-
somy 21 [2], trisomy 13 [3], trisomy 18 [4] and sex chromosome abnormalities
that included monosomy X [5], XXY [6] and XXX [7]. Several of these impor-
tant observations were followed by breakthroughs in the technological aspects of
cell cultures, which had been a stumbling block in routinely studying human
chromosomes. Two independent investigators, Nowell [8] and Moorehead et al.
[9], described a simple method of cell culture to study human chromosomes, thus
paving the way for the clinical cytogenetics revolution.
2.1 BASIC MORPHOLOGY
Each human chromosome consists of two arms joined by a “centromere”. The
two arms are termed p for the usually shorter arm and q for the longer arm.
Centromeres are essential structures where the mitotic spindles attach during cell
division. The centromere is a complex structure with mostly repetitive DNA that is
associated with a trilaminar plate structure called the “kinetochore”. Microtubules
attach to the kinetochore and help in directing the chromosome movements along
the spindle [10]. While the kinetochore structure and many centromere proteins
that are essential for the spindle fiber attachment are conserved during evolution,
the centromeric DNA sequences are not conserved among the eukarotyic organisms.
In humans the centromeric DNA consists of large blocks of middle-repetitive
DNA known as alpha satellite DNA that is AT (adenine, thymidine)-rich. These
repeats consist of a basic monomeric sequence of 170 bp arranged in a head to tail
manner. The total length of centromeric DNA varies from chromosome to
11
H. E. Wyandt et al. (eds.), Atlas of Human Chromosome Heteromorphisms
© Springer Science+Business Media Dordrecht 2004
, ATLAS OF HUMAN CHROMOSOME HETEROMORPHISMS
chromosome, as does the sequence of the 170 bp monomer between and within the
chromosomes [11–13]. Several studies with in situ hybridization, and the discovery
of the centromeric protein B (CENP-B), suggested that alpha satellite DNA consti-
tuted the centromere [12]. However, later studies have shown that associated DNA
sequences do not always function as centromeres and are not conserved during cen-
tromere evolution [14–16]. Recent studies of marker chromosomes that are mitoti-
cally stable with no apparent alpha satellite DNA, in humans and Drosophila,
further strengthen the proposal that alpha satellite DNA may not be necessary for
centromeric function [17–21]. The structure of the centromere and the role of cen-
tromeric DNA remain not fully understood.
Depending on the location of the centromere, human chromosomes can be clas-
sified into three groups. When the centromere is located in the middle with both
arms being more or less equal in length, the chromosome is called “metacentric”.
In the human karyotype, chromosomes 1, 3, 16, 19 and 20 are metacentric or near-
metacentric. When the centromere is located off-center with one arm longer than
the other, the chromosome is called “submetacentric”. Chromosomes 2, 4, 5, 6–12,
X, 17 and 18 are submetacentric. When the centromere is almost at the end of the
chromosome with one arm markedly smaller than the other, the chromosome is
called “acrocentric”. Chromosomes 13, 14, 15, 21 and 22 are acrocentric and are
distinguished by the presence of satellites or secondary constrictions. Secondary
constrictions are unstained regions or gaps in the short arms that contain nucleo-
lar organizing genes (NORs) [12,22]. Such secondary constrictions commonly
separate a small segment of chromosome called a “satellite” from the short arm.
In such cases the secondary constriction is referred to as a “satellite stalk” since it
connects the satellite to the chromosome short arm. Secondary constrictions are
also a distinctive feature of the pericentromeric regions of chromosome 1, 9 and
16. These constrictions do not contain NOR’s but consist of repetitive DNA
sequences that contribute to “constitutive heterochromatin” surrounding each
human centromere [23]. They stain dark with C-banding and show considerable
variation in length. Sometimes they are shorter than the average (e.g. 1qh⫺, 9qh⫺
and 16qh⫺; the h refers to heterochromatin) and sometimes many times longer
than the average (e.g. 1qh⫹, 9qh⫹ and 16qh⫹). Since repetitive DNA sequences
in these regions are not normally transcribed, variation in the content of this DNA
is considered to be a clinically insignificant, normal heteromorphism [24,25].
The chromosome ends contain special structures called “telomeres”. Telomeres
provide stability to the chromosomes by stabilizing the linear ends of DNA mole-
cules and are essential structures for maintaining the integrity of chromosomes.
They contain the simple DNA sequence, TTAGGG [26], repeated many times up
to 10 kb at the end of each chromosome arm. Unlike centromere sequences, this
simple telomere repeat is conserved throughout evolution. An RNA-containing
enzyme, “telomerase”, adds new repeat units to the ends of chromosomes to main-
tain telomere length. Over time, decreased telomerase function is thought to result
in progressive shortening of telomeres leading to senescence and cell death [27].
2.2 NOMENCLATURE
From the beginning it was recognized that standardization was needed in describ-
ing human chromosomes. To this end, several prominent investigators met in
12
, STUDYING HUMAN CHROMOSOMES AND NOMENCLATURE
Denver in 1960 [28] at the invitation of Dr T. T. Puck. The group, led by Dr C.
E. Ford, reached a consensus in the formulation of a common system. The auto-
somes were serially numbered, 1 to 22, in the order of descending length. The sex
chromosomes continued to be referred to as the X and Y. Also, the autosomes
were classified into seven distinct groups, with chromosomes within a group
arranged in descending order of length (Table 2.1). In order to arrange the chro-
mosomes in a karyotype, three primary measurements were used: the total length
of the chromosome relative to the total length of the haploid set with the X chro-
mosome; the arm ratio, the length of the long arm relative to the length of short
arm; and centromeric index, expressed as the ratio of length of short arm to the
length of the entire chromosome. In 1963, at the London Conference, the previ-
ously identified seven groups of chromosomes were designated by the letters A
to G, and the secondary constrictions were recognized.
Significant changes in nomenclature were again made after the discovery of
Q-banding by Caspersson et al. [29]. With the ability to identify each individual
chromosome based on banding patterns, it became essential to incorporate the
latest developments into the existing nomenclature resulting in the “Paris
Conference (1971)” [30] and its supplement [31]. The major highlights of these
documents are: (1) introduction of mosaicism and chimerism, (2) designation of
chromosome bands, (3) codes for describing the various banding methods and
(4) designation of heteromorphic variants. After Caspersson et al. showed the
presence of alternating dark and bright fluorescence patterns called bands on chro-
mosomes, the Paris Conference document published a diagrammatic representa-
tion of the banding patterns of each chromosome called an “ideogram” (Fig. 2.1).
In order to identify individual bands a distinct system of nomenclature was used.
Table 2.1 Architecture of human chromosomes (adapted from Paris Conference, 1971) [30]
Group 1–3 Large chromosomes in terms of length with centromeres located at
approximate center. Based on the length, it is easier to distinguish* these
three chromosomes.
Group 4–5 Large sub-metacentric chromosomes with very short short arms. It is difficult
to distinguish between chromosome 4 and 5 without banding,* but
chromosome 4 is slightly longer than chromosome 5.
Group 6–12 The most difficult group to distinguish.* All are sub-metacentric
chromosomes of medium length. The X-chromosome is included in this
group because of its length and architecture.
Group 13–15 Medium-sized chromosomes with centromeres at one end (acrocentric). They
are easy to distinguish from other groups but difficult to distinguish within
the group.* All of them may show satellites with considerable variation in
length and size of satellites.
Group 16–18 Short chromosomes with either metacentric (chromosome 16) or
sub-metacentric chromosomes (chromosomes 17 and 18).
Group 19–20 Short metacentric chromosomes. Often can be confused with
chromosome 16.*
Group 21–22 Very short, acrocentric chromosomes, easy to recognize by their size, but
difficult to distinguish from each other.* Chromosome 22 is actually longer
than chromosome 21. The Y chromosome is often included in this group
because of its morphological similarity.
*These comments refer to unbanded chromosome preparations. In banded preparations of adequate
quality, all pairs can be easily distinguished, one pair from another.
13
Methods of Studying Human
Chromosomes and Nomenclature.
The Normal Human Karyotype
GOPALRAO V.N. VELAGALETI AND VIJAY S. TONK
Ever since the elucidation of the correct human chromosome number (2n ⫽ 46)
by Tijo and Levan [1], clinical cytogenetics has become an important branch of
medical genetics. It was natural that this epoch-making discovery was soon fol-
lowed by discovery of various numerical chromosomal abnormalities such as tri-
somy 21 [2], trisomy 13 [3], trisomy 18 [4] and sex chromosome abnormalities
that included monosomy X [5], XXY [6] and XXX [7]. Several of these impor-
tant observations were followed by breakthroughs in the technological aspects of
cell cultures, which had been a stumbling block in routinely studying human
chromosomes. Two independent investigators, Nowell [8] and Moorehead et al.
[9], described a simple method of cell culture to study human chromosomes, thus
paving the way for the clinical cytogenetics revolution.
2.1 BASIC MORPHOLOGY
Each human chromosome consists of two arms joined by a “centromere”. The
two arms are termed p for the usually shorter arm and q for the longer arm.
Centromeres are essential structures where the mitotic spindles attach during cell
division. The centromere is a complex structure with mostly repetitive DNA that is
associated with a trilaminar plate structure called the “kinetochore”. Microtubules
attach to the kinetochore and help in directing the chromosome movements along
the spindle [10]. While the kinetochore structure and many centromere proteins
that are essential for the spindle fiber attachment are conserved during evolution,
the centromeric DNA sequences are not conserved among the eukarotyic organisms.
In humans the centromeric DNA consists of large blocks of middle-repetitive
DNA known as alpha satellite DNA that is AT (adenine, thymidine)-rich. These
repeats consist of a basic monomeric sequence of 170 bp arranged in a head to tail
manner. The total length of centromeric DNA varies from chromosome to
11
H. E. Wyandt et al. (eds.), Atlas of Human Chromosome Heteromorphisms
© Springer Science+Business Media Dordrecht 2004
, ATLAS OF HUMAN CHROMOSOME HETEROMORPHISMS
chromosome, as does the sequence of the 170 bp monomer between and within the
chromosomes [11–13]. Several studies with in situ hybridization, and the discovery
of the centromeric protein B (CENP-B), suggested that alpha satellite DNA consti-
tuted the centromere [12]. However, later studies have shown that associated DNA
sequences do not always function as centromeres and are not conserved during cen-
tromere evolution [14–16]. Recent studies of marker chromosomes that are mitoti-
cally stable with no apparent alpha satellite DNA, in humans and Drosophila,
further strengthen the proposal that alpha satellite DNA may not be necessary for
centromeric function [17–21]. The structure of the centromere and the role of cen-
tromeric DNA remain not fully understood.
Depending on the location of the centromere, human chromosomes can be clas-
sified into three groups. When the centromere is located in the middle with both
arms being more or less equal in length, the chromosome is called “metacentric”.
In the human karyotype, chromosomes 1, 3, 16, 19 and 20 are metacentric or near-
metacentric. When the centromere is located off-center with one arm longer than
the other, the chromosome is called “submetacentric”. Chromosomes 2, 4, 5, 6–12,
X, 17 and 18 are submetacentric. When the centromere is almost at the end of the
chromosome with one arm markedly smaller than the other, the chromosome is
called “acrocentric”. Chromosomes 13, 14, 15, 21 and 22 are acrocentric and are
distinguished by the presence of satellites or secondary constrictions. Secondary
constrictions are unstained regions or gaps in the short arms that contain nucleo-
lar organizing genes (NORs) [12,22]. Such secondary constrictions commonly
separate a small segment of chromosome called a “satellite” from the short arm.
In such cases the secondary constriction is referred to as a “satellite stalk” since it
connects the satellite to the chromosome short arm. Secondary constrictions are
also a distinctive feature of the pericentromeric regions of chromosome 1, 9 and
16. These constrictions do not contain NOR’s but consist of repetitive DNA
sequences that contribute to “constitutive heterochromatin” surrounding each
human centromere [23]. They stain dark with C-banding and show considerable
variation in length. Sometimes they are shorter than the average (e.g. 1qh⫺, 9qh⫺
and 16qh⫺; the h refers to heterochromatin) and sometimes many times longer
than the average (e.g. 1qh⫹, 9qh⫹ and 16qh⫹). Since repetitive DNA sequences
in these regions are not normally transcribed, variation in the content of this DNA
is considered to be a clinically insignificant, normal heteromorphism [24,25].
The chromosome ends contain special structures called “telomeres”. Telomeres
provide stability to the chromosomes by stabilizing the linear ends of DNA mole-
cules and are essential structures for maintaining the integrity of chromosomes.
They contain the simple DNA sequence, TTAGGG [26], repeated many times up
to 10 kb at the end of each chromosome arm. Unlike centromere sequences, this
simple telomere repeat is conserved throughout evolution. An RNA-containing
enzyme, “telomerase”, adds new repeat units to the ends of chromosomes to main-
tain telomere length. Over time, decreased telomerase function is thought to result
in progressive shortening of telomeres leading to senescence and cell death [27].
2.2 NOMENCLATURE
From the beginning it was recognized that standardization was needed in describ-
ing human chromosomes. To this end, several prominent investigators met in
12
, STUDYING HUMAN CHROMOSOMES AND NOMENCLATURE
Denver in 1960 [28] at the invitation of Dr T. T. Puck. The group, led by Dr C.
E. Ford, reached a consensus in the formulation of a common system. The auto-
somes were serially numbered, 1 to 22, in the order of descending length. The sex
chromosomes continued to be referred to as the X and Y. Also, the autosomes
were classified into seven distinct groups, with chromosomes within a group
arranged in descending order of length (Table 2.1). In order to arrange the chro-
mosomes in a karyotype, three primary measurements were used: the total length
of the chromosome relative to the total length of the haploid set with the X chro-
mosome; the arm ratio, the length of the long arm relative to the length of short
arm; and centromeric index, expressed as the ratio of length of short arm to the
length of the entire chromosome. In 1963, at the London Conference, the previ-
ously identified seven groups of chromosomes were designated by the letters A
to G, and the secondary constrictions were recognized.
Significant changes in nomenclature were again made after the discovery of
Q-banding by Caspersson et al. [29]. With the ability to identify each individual
chromosome based on banding patterns, it became essential to incorporate the
latest developments into the existing nomenclature resulting in the “Paris
Conference (1971)” [30] and its supplement [31]. The major highlights of these
documents are: (1) introduction of mosaicism and chimerism, (2) designation of
chromosome bands, (3) codes for describing the various banding methods and
(4) designation of heteromorphic variants. After Caspersson et al. showed the
presence of alternating dark and bright fluorescence patterns called bands on chro-
mosomes, the Paris Conference document published a diagrammatic representa-
tion of the banding patterns of each chromosome called an “ideogram” (Fig. 2.1).
In order to identify individual bands a distinct system of nomenclature was used.
Table 2.1 Architecture of human chromosomes (adapted from Paris Conference, 1971) [30]
Group 1–3 Large chromosomes in terms of length with centromeres located at
approximate center. Based on the length, it is easier to distinguish* these
three chromosomes.
Group 4–5 Large sub-metacentric chromosomes with very short short arms. It is difficult
to distinguish between chromosome 4 and 5 without banding,* but
chromosome 4 is slightly longer than chromosome 5.
Group 6–12 The most difficult group to distinguish.* All are sub-metacentric
chromosomes of medium length. The X-chromosome is included in this
group because of its length and architecture.
Group 13–15 Medium-sized chromosomes with centromeres at one end (acrocentric). They
are easy to distinguish from other groups but difficult to distinguish within
the group.* All of them may show satellites with considerable variation in
length and size of satellites.
Group 16–18 Short chromosomes with either metacentric (chromosome 16) or
sub-metacentric chromosomes (chromosomes 17 and 18).
Group 19–20 Short metacentric chromosomes. Often can be confused with
chromosome 16.*
Group 21–22 Very short, acrocentric chromosomes, easy to recognize by their size, but
difficult to distinguish from each other.* Chromosome 22 is actually longer
than chromosome 21. The Y chromosome is often included in this group
because of its morphological similarity.
*These comments refer to unbanded chromosome preparations. In banded preparations of adequate
quality, all pairs can be easily distinguished, one pair from another.
13
