CDC’s 2003 Science Ambassador Program Chromosomal Karyotypes Dawn Adams Cytogenetics CDC’s 2003 Science Ambassador Program In this presentation, we will be looking into the field of cytogenetics, the study of genetics dealing with chromosomes and their genetic implications. In particular, we will be exploring karyotypes. Karyotypes are commonly used to investigate chromosomal number and banding, and are particularly helpful in looking for chromosomal abnormalities. (1)

Overview I. Chromosomes A. Definition B. Structure C. Identification II. Karyotypes B. Methods C. Staining D. Importance In this presentation, we will be learning about chromosomes, karyotypes, and two methods for obtaining a sample for a karyotype. We will start out by talking about the definition and structural description of chromosomes. We will then talk about a chromosomal sample viewed at metaphase called a karyotype. Finally, we will discuss amniocentesis and chorionic villus sampling, which are two techniques used in conjunction with karyotypes to investigate chromosomal abnormalities in a fetus.

Chromosomes Definition Genetic structures of cells containing DNA Identification Each chromosome has a characteristic length and banding pattern Chromosomes are structures in the nucleus of cells that contain genetic material or DNA. This DNA bears in its nucleotide sequence the linear array of genes. Each chromosome has a characteristic length and banding pattern. (1)

The breakdown of a Chromosome Each autosome is numbered from 1-22, sex chromosomes either X or Y p arm (short arm) Each autosome, or a chromosome not involved in determining sex, is numbered from 1 to 22 in order of decreasing length. There are two identical chromosomes for these chromosomes 1-22. These chromosomes are said to be homologous pairs. In other words, they have identical size, structure, and a similar nucleotide sequence. The sex chromosomes X and Y are not homologues. The X chromosome is longer than the Y chromosome. On an individual level, the short arm of the chromosome is labeled the p arm, while the long arm is labeled the q arm. The connection point of the two autosomes is called the centromere. (1) q arm (long arm) Centromere

Chromosome Labeling Example - 1q2.4 Each arm divided into sub-regions and identified by a number Each sub-region divided into bands identified with a number Chromosome is identified with a number ranging 1-22, or X and Y Example - 1q2.4 The first chromosome, long arm, second region of the chromosome, the fourth band of that sub-region Multiple parts of a chromosome are labeled. First, the chromosome itself is numbered. The autosomes, or chromosomes that are not involved in determining sex, are labeled 1-22 based on size from longest to shortest. Chromosomes that determine sex are labeled X or Y. Then, the chromosome is divided into the p and q arm regions. Each arm is then divided into sub-regions which are identified with a number. Finally, each sub-region is divided into bands identified with a number. (1)

A Karyotype Definition 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y Definition A photographic arrangement of a complete set of chromosomes of a cell or organism A karyotype is a photographic arrangement of homologous pairs of a complete set of chromosomes. The illustration on the left shows an example of a karyotype (note that real karyotypes can be seen on the web practice in step 2). Notice that there are 22 pairs that decrease in size as the numerical identification get bigger. X and Y chromosomes are also displayed and therefore the sex status can be determined. (1)

Obtaining a Sample Fetal samples for karyotypes are commonly obtained in two ways Amniocentesis – sample taken from the fluid of the amniotic sac Chorionic Villus Sampling – sample taken from the fetal tissue that forms part of the placenta Karyotyping is a technique that can be used at any age. There are many ways to obtain a sample, however we will focus on two samples commonly used to obtain fetal samples. These fetal karyotypes can help determine chromosomal abnormalities before a baby is even born. Amniocentesis Amniocentesis is a process done to obtain a sample from an infant to be karotyped. It is normally considered in situations in which parents are at risk of having an infant with a genetic disorder. This includes situations of advanced maternal age, parents who have had a previous child with chromosomal abnormalities and parents with the presence of a chromosomal translocation or rearrangement. More than 100 biochemical disorders can be tested through amniocentesis. (2) During this procedure, amniotic fluid, which surrounds the baby and provides protection, is taken from the amniotic sac by a long needle inserted into the woman’s abdomen. The body will make more amniotic fluid to replace the fluid removed in the procedure. (2) Chorionic Villus Sampling Chorionic villus sampling is another procedure used to help determine genetic disorders present in the fetus. While this procedure is recommended for women with the same risk factors as in amniocentesis, it is more specialized than amniocentesis and can be done earlier in pregnancy (during about 10-12 weeks of pregnancy). (3) During this procedure, a small, flexible catheter is inserted through the vagina or abdomen into the uterus and is guided by ultrasound images. A sample is taken from the chorionic villus, the fetal tissue that forms part of the placenta, by suction. (3)

Obtaining a Karyotype Chromosomes are stained for easy visualization Light microscope used to view chromosomes in metaphase of mitosis Chromosomes arranged into homologous pairs based on size and banding patterns Mitosis is a form of cell division that produces two identical cells. Each chromosome is aligned along the spindle equator during the metaphase of mitosis, making the pairs easy to see. Chromosomes are stained and, by using a light microscope, a scientist can then observe these chromosomes and arrange them in size order. Homologous pairs, or chromosomes with identical size, structure, and similar nucleotide sequence, are placed next to each other. (1)

Staining Banding patterns can be visually identified on chromosomes after staining. Traditional Types G-Banding – Giemsa stain Q-Banding – Fluorescent stain R-Banding – Reverse Giemsa stain New Type Fluorescence In Situ Hybridization techniques Staining procedures for karyotypes produce patterns of bands that are unique for each chromosome. Comparison of these band can help to match homologous pairs in a karyotype. Types of staining include G-banding which involves Giemsa stain, Q-banding, which involves fluorescent staining and R-banding which involves a reverse Giemsa stain, in which the complements of G-banding can be visualized. (4) Procedure of staining G-Banding : Chromosomes are treated with an enzyme (trysin) to digest some chromosomal proteins. Chromosomes are then exposed to Giemsa stain, which consists of a mixture of dyes and results in darkly stained G bands which are visible under a microscope. (5) Q-Banding : Chromosomes are treated with quinacrine mustard and patterns are observed by placing the sample under a special type of ultraviolet light microscope. The chromosomes will show bright fluorescent bands. (6) R-Banding : Chromosomes are treated with acridine orange and observed with a light microscope. The result is a darkly stained centromere region of the chromosome. (7) It is important to note that new molecular techniques such as Fluorescence In Situ Hybridization (FISH) are now replacing some of the more traditional staining methods because of the limitations of the light microscope. These new techniques enable visualization of small duplications, deletions, or rearrangements that can no be seen with traditional cytogenetics. (8)

Importance of Karyotypes Karyotypes show the chromosomal makeup of an individual. Knowing the number of chromosomes is essential for identifying chromosomal variations that cause genetic disorders. A karyotype allows us to determine the chromosome makeup of an individual. It can show if a person has 46 chromosomes or if a person has too many or too few. These latter two cases are chromosomal variations that can cause a range of disorders in humans. Chromosomal variants and the syndromes that arise from these variations will be discussed in the next PowerPoint presentation.

References Fairbanks, D. J., Anderson, W. R. Genetics: The Continuity of Life. Pacific Grove, CA: Brooks/Cole Publishing Company; 1999. NIH. Amniocentesis [online]. 2004. [cited 2004 Feb 6]. Available from URL: http://www.nlm.nih.gov/medlineplus/ency/article/003921.htm. NIH. Chorionic villus sampling [online]. 2004. [cited 2004 Feb 6]. Available from URL: http://www.nlm.nih.gov/ medlineplus/ency/article/003406.htm. Campbell, N. A. Biology. 3rd ed. Redwood City, CA: The Benjamin/Cummings Publishing Company, Inc.; 1993.

References (continued) On-line medical dictionary. G-banding: Banding pattern. 1997. [cited 2004 Feb 6]. Available from URL: http://cancerweb.ncl.ac.uk/cgi-bin/omd?G-banding. On-line medical dictionary. Q-banding. 2000. [cited 2004 Feb 6]. Available from URL: http://cancerweb.ncl.ac.uk/cgi-bin/omd?query=q-banding. On-line medical dictionary. R-banding stain. 2000. [cited 2004 Feb 6]. Available from URL: http://cancerweb.ncl.ac.uk/cgi-bin/omd?R-banding+stain. National Human Genome Research Institute, Fluorescence In Situ Hybridization (FISH). 2004 [cited 2004 Feb 6]. Available from URL: http://www.genome.gov/10000206.