HUMAN CHROMOSOMES Normal human somatic cells contain a diploid number of chromosomes (2n=46), so there are 23 pairs of chromosomes: - 22 pairs are identical in man and women and are called autosomes; - 1 pair of sex chromosomes: XY in men and XX women. The maternally and paternally derived chromosomes present in a diploid cell that contain equivalent genetic information, are similar in morphology, and pair during meiosis are called homologous chromosomes. The mature sexual sells the gametes contain a haploid number of chromosomes (n=23); the sperm cells contain 22+X (23,X) or 22+Y (23,Y); the egg cells contain 22+X (23,X). A display of the metaphase chromosomes of a somatic cell of an individual, based on their number, shape, size and other landmarks (secondary constriction, satellites, bands) is called karyotype. The karyotype is normally shown as a photomicrograph of the chromosomes arranged in a standard way. Photographs of chromosome pairs are aligned to provide a visual representation of the organism's chromosomal constitution. The process of preparing a photomicrograph is known as karyotyping. Individual chromosomes are identified by chromosome banding. A typical metaphase chromosome consists of two sister chromatids connected by centromere (primary constriction) - the point where the two chromatids remain attached, but also containing the kinetochore, the point of spindle attachment. Each chromatid contains two arms: short (p) and long (q), separated by primary constriction. The ends of chromatids are called telomeres, which contain repetitions of TTAGGG sequence. They prevent the fusion of chromosomes, protect the DNA from the action of exonucleases and are involved in full replication of DNA (through the activity of telomerase). Their biological role is thus providing the stability and maintaining the integrity of chromosomes. Some chromosomes contain less condensed and less stained fragments called secondary constriction. There are some morphological criteria based on chromosome s size and configuration used for identification of chromosomes: - The chromosomal length. Usually the absolute length (in micrometers) or relative length is used. The relative length is calculated using the formula: The lenght of studied chromosome X 100. The total length of all chromosome s from a haploid cell The chromosomes may be large, medium and small. Another classification criterion is based on the position of centromere, which is characterized by centromere index (arm length ratio), calculated p through the formula: Ic X100. p q Using this criterion, human chromosomes are divided in: metacentric, submetacentric, and acrocentric.
Human Chromosomes metacentric: Ic = 46 49 % submetacentric: Ic = 31 45 % acrocentric: Ic = 17 30 % Methods of Chromosome Analysis Chromosomes could be analyzed through cytogenetic and molecular cytogenetic techniques. Chromosomes can best be studied at metaphase, when chromosome condensation is maximal and all chromosomes are in one plane. To visualize metaphase chromosomes, they must first be fixed on a slide, and then stained, either uniformly with a dye such as orcein, or for more resolution and detail, with banding techniques after gentle denaturing treatments. The chromosomes from actively dividing tissues may be examined directly (marrow cells, tumors). The other cells should be cultured before studying. There are two types of cell cultures, depending on examined tissues: - Short time cultures for blood cells; - Long time culture for skin, amniotic or solid tumor cells. The steps of blood cell (lymphocytes) culture: - Collecting of blood in aseptic condition; - Culturing in sterile vials containing special media at 37 0 C, during 72 hours. For mitosis induction a special mitogen phytohaemagglutinin is added; - Blocking of mitosis at metaphase using colchicines or colcemid; - Adding of hypotonic solution, which disperses the chromosomes in the cells; - Adding of fixative (3:1, alcohol:acetic acid), which kills the cells and keeps the natural shape of chromosomes; - Spreading of cells on a slide; - Staining, using an appropriate dye; - Examination, using a microscope; - Photographs of chromosome pairs are aligned to provide a visual representation of the chromosomal constitution. Uniform Staining The chromosomes are stained without special treatment at the metaphase stage. Usually Giemsa dye or orcein are used for staining. This method provides information only about the number and morphology of chromosomes. The chromosomes could be grouped on the basis of their relative sizes and the relative lengths of their two arms, i.e. the positions of their centromeres. Chromosomal Banding If chromosomes are treated briefly with protease before staining then each chromosome has a characteristic-banding pattern. Different dyes provide different patterns of banding. The two main banding techniques used are Giemsa banding (G banding) and reverse banding (R banding), which are grossly complementary and enable the detection of some 150200 bands generally agreed upon and officially recognized by the ISCN (International System for Human Cytogenetic Nomenclature). A still higher resolution can be obtained by synchronizing cell division and blocking the 2
Human Chromosomes chromosomes in a prometaphase stage, so that the chromosomes are more elongated and may show up to 600 and even 1000 bands. Different Approaches of Chromosome Banding Type Dye Band Origin Practical Role G-banding Giemsa The positive bands - heterochromatin The negative bands euchromatin Exact identification of chromosomes, identification structural abnormalities Q-banding Quinacrin (fluorescent) The positive bands - heterochromatin The negative bands euchromatin Exact identification of chromosomes, identification structural abnormalities R-banding (revers) Giemsa or fluorescent dyes The positive bands - euchromatin The negative bands heterochromatin Exact identification of chromosomes, identification structural abnormalities Exact identification of chromosomes C-banding (centromere) Giemsa or fluorescent dyes The positive bands heterochromatin which surround centromere Exact identification of chromosomes T-banding (telomere) Giemsa or fluorescent dyes The positive bands heterochromatin in telomeres FISH (Fluorescent in situ Hybridization) This method is based on complementary binding of single-stranded DNA labeled with fluorescent dye (hybridization probe) to denatured chromosomes. Hybridization allows visualization of a DNA fragment of as little as 1-2 kb (but more usually 40-50 kb) at an efficiency approaching 100%. It is useful for identifying the position of a gene in the chromosome, as well as chromosomal aberration. Chromosomal Painting This technique represents a variety of FISH, when complex probes made from entire chromosomes are used. Different parts of the chromosome are colored specifically. In this way the presence of a chromosome can be ascertained, as well as different rearrangements. The principle of FISH 3
Human Chromosomes Alternatively, each chromosome may be painted in a specific color: Spectral Karyotyping (SKY) and Multiplex Fluorescence in situ Hybridization (M-FISH). SKY and M-FISH are molecular cytogenetic techniques that permit the simultaneous visualization of all human chromosomes in different colors, considerably facilitating karyotype analysis. SKY/M-FISH is particularly useful in: mapping of chromosomal breakpoints; detection of subtle translocations, characterization of complex rearrangements. Comparative Genomic Hybridization (CGH) Comparative genomic hybridization (CGH) is a fluorescent molecular cytogenetic technique that identifies DNA gains, losses, and amplifications, mapping these variations to normal metaphase chromosomes. It is a powerful tool for screening chromosomal copy number changes in tumor genomes and has the advantage of analyzing entire genomes within a single experiment. It is particularly applicable to the study of tumors which do not yield sufficient metaphases for cytogenetic analysis. Autoradiography. Is based on introducing of radioactive labeled nucleotides (dttp, treated with tritium) during replication in vivo. It assures identification of newly synthesized strands of DNA. So it is possible to identify which chromosome or part of chromosome replicates early or lately during S-phase (e.g. chromosomes 8, 13, 18, 21 and one of X in women replicate last). Classification of Human Chromosomes Based on different quantity criteria (length and centromere index) and quality criteria all human chromosomes are divided in 7 groups, marked A, B, C, D, E, F, G. Group Size, Shape Chromosomes Landmarks Notes A large, metacentric 1 3 1qh+ the largest chr. B large, submetacentric 4, 5 C medium, submetacentric 6 12 and X 9qh+ 16 chr. in and 15 in D medium, acrocentric 13 15 ps+; ph+ h contain nucleolar organizing genes E medium, metacentric 16 16qh+ small, submetacentric 17, 18 F small, metacentric 19, 20. G small, acrocentric 21, 22 Y ph+; ps+ ph form NOR (contain rrna) Yqh+ 4 chr. in and 5 chr. in The satellites represent short fragments of heterochromatin at the end of chromosome, separated by secondary constriction. They are present in chromosomes 13, 14, 15, 21, 22 (all acrocentric chromosomes, except Y). In the short arms of acrocentric chromosomes, secondary constrictions are associated with the nucleolar organizing regions (NOR). The secondary constrictions represent less condensed and less stained fragments of chromosomes. In addition to acrocentric chromosomes, they are usual for long arms of chromosomes 1, 9, 16, 19. These constrictions do not contain NOR, but consist of repetitive DNA sequences. These regions show considerable variations in length (ex: shorter than the average, 1qh-, or longer than the average, 16qh+), which are considered clinically insignificant and represent normal polymorphism (heteromorphism). Length Position of centromere Metacentric Submetacentric Acrocentric Large A 1, 2, 3 B 4, 5 - Medium E 16 С 6, 7, 8, 9, 10, 11, 12, X D 13, 14, 15 Small F 19, 20 E 17, 18 G 21, 22, Y 4
Human Chromosomes An idealized human karyotype after G-banding (46,XY) Karyotype: Chromosomal Formulas Some landmarks may be distinguished in each chromosome: The arms may contain one or some regions separated by secondary constriction or prominent, large bands. The regions are marked with numbers, beginning from the centromere. Different chromosomes contain diverse number of regions (e.g. chromosome 1 contains 3 regions in p-arm and 4 regions in q-arm). Each region consists of bands, which are marked with numbers in direction from centromere to telomere. The metaphase chromosomes contain 400 500 bands. At the prometaphase stage (short stage between prophase and metaphase), when the chromosomes are less condensed, the bands may be divided in some subbands. The prometaphase chromosomes contain 1800 2000 subbands. This stage is used for high resolution karyotyping. 5
The normal karyotype formulas: the number of chromosomes, coma, sex chromosomes 46,XX for women ( ) 46,XY for men ( ) Human Chromosomes The formulas of karyotypes containing numeric errors: Errors of sex chromosomes - the number of chromosomes, comma, sex chromosomes 47,XXX a woman with an extra X chromosome 47,XXY a man with an extra X chromosome 47,XYY a man with an extra Y chromosome 45,X a woman missing a sex chromosome Errors of autosomes - the number of chromosomes, comma, sex chromosomes, comma, plus (minus) chromosome 47,XX,+13 a woman with an extra 13 chromosome 47,XY,+21 a man with an extra 21 chromosome The formulas of karyotypes containing structural abnormalities: The structural aberrations are marked with special symbols. Symbol Name Characterization del deletion Loss of a segment of the genetic material from a chromosome dup duplication A chromosome segment occurs more than once inv inversion The order of several genes is reversed from the normal order i (iso) isochromosome A chromosome with two identical arms r ring Ring chromosome rob Robertsonian translocation The long arms of two acrocentric chromosomes become joined to a common centromere t translocation Interchange of parts between nonhomologous chromosomes 46,XX,del(1)(q11q13) a woman, 46 chromosomes, deletion of a segment between band 1 and band 3 of region 1 of short arm of 1 st chromosome. 46,XY,inv(2)(p21q31) a man, 46 chromosomes, inversion of the segment between region 2, band 1 in short arm and region 3 band 1 in long arm of chromosome 2. 46,XX,dup(1)(q21.1q21.3) a woman, 46 chromosomes, duplication of a segment in long arm of chromosome 1 between region 2, band 1, subband 1 and region 2, band 1, subband 3 46,X,i(Xq) [or 46,X,iso(Xq)] a women, 46 chromosomes, one of chromosomes X contains only long arms. Variations in chromosome number and structure in persons with normal phenotype In women after 60 years old ~ 7% of cells may lose one of chromosome X and become 45,X. In men after 70 years old ~ 2% of cells may lose the chromosome Y and become 45,X. Some chromosomes (1, 9, 16, Y) contain a very long secondary constriction. Sometimes satellites may be observed in chromosomes 17, 18. The bands width (Q, G, C) may differ in chromosomes of different origin. These peculiarities offer possibility to identify the origin of chromosomes (maternal or paternal) 6
HUMAN SEX CHROMOSOMES Human Chromosomes In humans, the sex chromosomes are labeled X and Y. Females have two X chromosomes and males have one X and one Y chromosome. All the eggs produced during meiosis have an X chromosome (23,X). Half of the sperm produced by a male contain an X chromosome (23,X) and the other half have a Y chromosome (23,Y). Thus, sperm determine the sex of the offspring. If the egg is fertilized by a sperm with an X chromosome, the zygote develops into a female. If the sperm contains a Y chromosome, the offspring is a male. There is a difference between the sex chromosomes. While the X chromosome is relatively large (approximately 6% of nuclear DNA), the Y chromosome is quite small, and only a few genes have been assigned to it. X-chromosome inactivation In 1961, the British geneticist Mary Lyon proposed that the X chromosomes in somatic cells of mature females are of two types. One is active and expresses its full complement of genetic information; the other is inactivated in some manner and does not serve as a source of genetic information. The biological meaning of suppression of the functional activity of one of two X chromosomes is the dose compensation, as in male karyotypes there is only one X chromosome present, and in female - two. Thus the genotypic possibilities of male and female karyotype are equalized. It is important that this inactivation occurs randomly, so that in early embryonic life (after 16 days) different cells may have alternative X chromosomes inactivated. So, in some cells, the X-linked genes inherited from the mother are expressed, whereas in other cells, the X-linked genes inherited from the father are active. The somatic tissues of females are thus said to be mosaic because they represent the contribution of genes from different X chromosomes. In each somatic cell the genes in only one X chromosome will be expressed, but the X chromosome that is genetically active will differ from cell to cell. The mosaicism has been observed in women who are heterozygous for an X-linked recessive mutation resulting in the absence of sweat glands; these women exhibit areas of skin in which sweat glands are present (these parts derived from embryonic cells with normal X chromosome active and the mutant inactive), and other areas of skin in which sweat glands are absent. The molecular basis for X chromosome inactivation is not completely understood. The process begins with activation of a gene called X-inactivation-specific transcript (XIST) on the long arm of the X chromosome (Xq13). XIST is expressed only in the inactive X chromosome and produces an RNA molecule (not translated into protein) that transmits the inactivation signal throughout the chromosome. The process involves physical reorganization of the DNA within the chromatin and also the addition of methyl groups to the DNA bases. 7
Barr Body Human Chromosomes One of the two X chromosomes in female cells is condensed in form of facultative heterochromatin during interphase and forms the Barr body. A Barr body is about 1 micrometer in diameter and is located at the periphery of the nuclear membrane. The number of Barr bodies is one less than the number of X chromosomes. Cells of normal females have one Barr body; cells of normal males have none. Individuals with two or more X chromosome have a number of Barr bodies equal to the number of X chromosomes minus one (that is, equal to the number of inactivated X chromosomes). For example, XXX individuals have two Barr bodies, XXXX individuals have three, and XXXXX individuals have four. Thus, an XYY male has no Barr bodies, and XXY or XXYY males have one Barr body. Determination of X-chromatin in mucosa cells Barr bodies can be determined most easily in buccal mucosa, hair roots and fibroblast cells. The normal positive range for sex chromatin bodies is 20-60 percent. Determination of sex chromatin in mucosa cells includes the following stages: 1) making the preparation; 2) microscope analysis of the preparation; 3) conclusions making. Epithelium cells of the mucosa from the internal part of a cheek serve as material. Before taking the scrape of the cells, the mouth should be rinsed with clean water. A scrape is made using a sterile scalpel. The scrape is spread over the object-plate, which is dipped into methyl alcohol for fixation. 10-15 minutes later the preparation is taken out of the alcohol, air-dried. Then one or two drops of acetoorceine are added on the preparation, covered with a cover-glass and the excess of dye is removed by blot. Microscopic examination of preparation follows. For microscope analysis, only the nuclei with chromatin appeared as oval or kidney-shaped bodies are taken into account. The bodies are usually localized by the inner surface of nuclear envelope. About 100-150 cells are analyzed; those containing Barr bodies are counted. The frequency of cells containing Barr bodies is calculated in %. Examination of Barr bodies is a rapid and convenient test. It is useful to: determine the sex in prenatal stage; determine the sex of a person with ambiguous genitalia, detect abnormal karyotypes such as Turner syndrome (45,X) and Klinefelter syndrome (polysomy X in male). 8
Y Chromosome Human Chromosomes The Y chromosome likely contains approximately 400 genes. Because only males have the Y chromosome, the genes on this chromosome tend to be involved in male sex determination and development. Sex is determined by the SRY (Sexdetermining Region Y) gene, located on the short arm, which is responsible for the development of a fetus into a male. SRY is thus a Y-linked gene, because it is found only on the Y chromosome. Other genes on the Y chromosome are important for male fertility. In case of a translocation of SRY gene to X chromosome a 46,XX testicular disorder of sex development occurs. This means that a fetus with two X chromosomes, one of which carrying the SRY gene, will develop as a male despite not having a Y chromosome. Deletions of genetic material in regions of the Y chromosome called azoospermia factor (AZF) usually cause Y chromosome infertility. Genes in these regions are believed to provide instructions for making proteins involved in sperm cell development, although the specific functions of these proteins are unknown. Many genes are unique to the Y chromosome, but genes in areas known as pseudoautosomal regions (PAR) are present on both sex chromosomes. As a result, men and women each have two functional copies of these genes. Many genes in the pseudoautosomal regions are essential for normal development. Y-chromatin Structure of Y chromosome Y-chromatin or F body is formed by 2/3 of q arm of the Y chromosome and can be observed microscopically as an intensive fluorescent staining body in the nucleus of interphase cells. Its size is about 0.25 µm and it is situated apart or is attached to nuclear membrane. The frequency of cells containing F bodies varies in different tissues of male organism. For example, it is 70-85% in fibroblasts and it is about 45% in sperm cells. The number of Y-chromatin bodies is equal with the number of Y-chromosomes. Thus, cells of normal females have no F bodies, cells of normal males have one F body, XYY males have two F bodies. Cells with is isochromosome Y containing two long arms will present one F-body twice bigger than normal (0.5 µm). F-body test is used for identification of the changes in the number of copies of Y chromosome. Such changes might include: 48,XXYY syndrome. Extra genetic material from the X chromosome interferes with male sexual development, preventing the testes from functioning normally and reducing the levels of testosterone (male hormone). A shortage of testosterone during puberty can lead to reduced facial and body hair, poor muscle development, low energy levels, and an increased risk for breast enlargement (gynecomastia). Dental problems are frequently seen with this condition; they include delayed appearance of the primary or secondary teeth, thin tooth enamel, crowded and/or misaligned teeth, and multiple cavities. Extra copies of genes from the pseudoautosomal region of the extra X and Y chromosome contribute to the signs and symptoms of 48,XXYY syndrome; however, the specific genes have not been identified. 9
Human Chromosomes 47,XYY syndrome, also called Jacob's syndrome or YY syndrome. Although males with this condition may be taller than average, this chromosomal change typically causes no unusual physical features. Most males with YY syndrome have normal sexual development and are able to father children. It is unclear why an extra copy of the Y chromosome is associated with tall stature, learning problems, and other features in some men. A small percentage of males with Jacob's syndrome are diagnosed with autistic spectrum disorders, which are developmental conditions that affect communication and social interaction. 10