Taxonomic Identification of Biological Organisms.

Instruction Manual

TABLE 1. Diploid chromosome numbers of various organisms.

The second major reason for interest in human chromosomes was a report in 1959 that cells from people with Down's syndrome (mongolism) contained an extra chromosome. This was the first known association between a chromosomal abnormality and human disease. It set off a search which has resulted in the discovery of a wide variety of chromosomal abnormalities ranging from interchanged chromosome sections with no clinical effects to minute changes that are lethal.

Most numerical chromosomal abnormalities arise from failure of a pair of homologous chromosomes to separate properly during meiosis (nondisjunction). As a result, one of the daughter cells has one or more extra chromosomes and the other is lacking an equivalent number. In humans a loss of chromosomal material is more lethal than a gain. Turner's syndrome (females with only one X chromosome) provides the only known example of humans surviving with less than the normal number of chromosomes.

There are some other disorders such as "cri du chat" syndrome (named from the observation that newborn infants with this disorder have an unusual cry which sounds like a kitten's meow) where a person can survive with part of a chromosome missing. Loss or gain of chromosomal material is frequently but not always associated with mental retardation. Chromosomal abnormalities probably account for about 10 to 15 percent of all cases of mental retardation.

It is important to note some of the basic differences between single gene disorders such as phenlyketonuria (an inability to metabolize a particular amino acid (phenylalanine)) which leads to severe mental retardation if untreated and chromosomal disorders such as Down's syndrome. One of the major functions of genes is to supply the proper amino acid sequence for assembling proteins. A useful analogy is to think of the chromosomes as serving as a dictionary for the cell. When we want to assemble the letters of the alphabet into a particular word, we look up the correct sequence in the dictionary. Similarly, each structural gene tells the cell the proper sequence in which to assemble the amino acids in order to make a particular protein molecule. Each normal human somatic cell contains 23 pairs of chromosomes, one member of each pair is from the father and the other from the mother. In essence, the diploid cell has two similar dictionaries, one from each parent. A disorder due to a single recessive gene is like having a misprint in one word of the dictionary. For such a disorder to appear, both parents must have at least one form of the gene (allele) which carries the wrong instructions. The Punnett square (Fig. 1) shows that if both parents are carriers of the disease (i.e., one allele carries the correct instructions, P, for a certain protein and the other allele carries the wrong instructions, p) there is one chance in four that the zygote will receive no correct instructions for the protein and will therefore develop the disease.

Figure 1. Punnett square showing the pattern of inheritance from parents carrying a recessive gene.

In some cases the amount of a particular protein produced appears to be proportional to the number of correct instructions. Thus, the normal person (PP) with two sets of correct instructions would be expected to have twice as much of this particular protein as the carrier (Pp) with only one set of correct instructions. Generally, the decrease found in the carrier does not result in any clinical symptoms although it can often be detected with special chemical tests. In other words, PP and Pp may appear- to be the same physically, but we can often distinguish between them chemically.

The chromosomal disorders differ from single gene disorders in that the parents rarely are carriers of any defect and the basic problem is too few or too many normal genes rather than an abnormal gene. Having an extra chromosome is like having an extra page in our dictionary. The additional instructions (genes) supplied by the extra chromosome would be expected to cause abnormally high production of some proteins. This may have a severe effect on the development of the zygote as can be seen by the fact-that the presence of an extra chromosome 21 (trisomy) causes the symptoms associated with Down's syndrome. Trisomy of the larger chromosomes, which presumably carry even more genes, is usually not compatible with life. The sex chromosomes are an exception as it is possible for a person to have extra sex chromosomes with relatively little known effect.

Many chromosomal abnormalities arise from errors occurring during cell division. The fact that Down's syndrome occurs about once in every 700 births suggests that these errors are not extremely rare. A number of such errors probably occur throughout our bodies each day. However, it is only those which occur during formation of gametes that are regularly observed since they can be passed on to the offspring.

Translocations represent a special type of chromosomal abnormality which may be transmitted through a family. In this situation, parts of two different chromosomes become joined. A person can carry a translocation and be otherwise normal but it may be difficult, or sometimes impossible, to produce gametes with the correct amount of genetic information.

Chromosome Structure

Human metaphase chromosomes have characteristic shapes as shown in the sample karyotype. In Figure 5 they appear to be double except at the region of constriction, the centromere. The various "V" and "X" shapes depend on the length of the chromosome and the position of the centromere. A "V" shape results when the centromere is near an end, and such chromosomes are called "acrocentrics". The "X" shaped chromosomes, with their centromeres in the middle, are called "metacentric". As shown in Figure 2 the short arm of a chromosome is referred to as "p" and the long arm as "q". Chromosomes can be classified according to their "centromeric index" which can be thought of as the percentage of the total length contributed by the short arm.

Karyotype Analysis

Preparation of a standard diagnostic karyotype involves cutting the individual chromosomes out of an enlarged photograph of a dividing cell (Figure 4, 6). Each chromosome is then matched with the other member of its pair and fixed onto its proper position on a standard karyotype form. The karyotype form organizes the chromosomes into 7 groups (A through G) based on size, shape, centromeric index, and banding pattern if available (Figure 5, 7). Any numerical or major structural abnormalities will be obvious at this stage.

Although karyotype analysis is useful for clinical diagnosis of certain diseases it is most often used in conjunction with amniocentesis (Figure 3) to determine the chromosomal make-up of a fetus before birth. It is now possible to diagnose many genetic diseases prenatally and this raises important ethical and moral questions regarding the termination of pregnancy involving an abnormal fetus.

Figure 3. Amniocentesis. A sample of amniotic fluid (mostly fetal urine and other sections) is taken by inserting a needle into the amniotic cavity during or about the 16th week of gestation. The fetal cells are separated from the fluid by centrifugation. The cells are then cultured so that a number of biochemical, enzymatic, and chromosomal analyses can be made.

Figure 4. Metaphase spread of unbanded chromosomes from a normal male (XY).

Photo Credit: Carolina Biological Supply Company 1975

Figure 5. Unbanded karyotype of a normal male subject. (Prepared from Figure 4)

Figure 6. Metaphase spread of banded chromosomes from a normal female (XX).

Photo Credit: Carolina Biological Supply Company.

Figure 7. Banded karyotype of a normal female subject: (Prepared from Figure 6)

Preparation of Chromosomes for Karyotype Analysis

Chromosomal preparations are most easily obtained from white blood cells (lymphocytes). The preparation is set up by placing a few drops of blood in a tube containing growth medium supplemented with phytohemagglutinin to stimulate the lymphocytes to divide. The tube is incubated at 37°C. After 72 hours colchicine is added and incubation is continued for another 1 1/2 hours. Colchicine will arrest dividing cells at metaphase.

At metaphase, each chromosome has replicated itself with the two strands joined at the centromere. If cell division had continued, the strands would have pulled apart and one strand would have entered each of the new daughter cells. Thus, the X-shaped structures seen in the photographs would have become two chromosomes if they had been allowed to separate.

The chromosomal preparation is centrifuged to separate the cells and then treated with a hypotonic solution to destroy the red cells and make the lymphocytes easier to rupture. The lymphocytes are then fixed in a mixture of methanol and glacial acetic acid. The fixed cells are dropped on a cold slide and air dried to rupture the cells and spread the chromosomes. The slide is washed and stained with Giemsa. Treatment of the chromosomes with enzymes or mild acid before staining causes a characteristic banding pattern for each chromosome. Such "banded" preparations allow more precise matching of chromosome pairs and identification of structural abnormalities. Compare the banded and unbanded karyotypes of Figure 5 and 7.

While the chromosomes are visible under low power (100X), counting and photography are routinely done under oil immersion (1000X). The chromosomes in at least 25 cells are usually counted to be reasonably certain of detecting any chromosomal abnormalities. One or two well-spread fields are then photographed for karyotyping.

Genetic Conditions Detectable by Karyotype Analysis

Cri-du-chat Syndrome (5p deletion) A chromosomal abnormality caused by deletion of all or part of the short arm of chromosome 5. The most distinctive characteristic of this syndrome is the infant's cry, which is identical to that of a mewing kitten. In addition, there are characteristic abnormalities which include a small head, moon-like face, and receding lower jaw. Affected individuals are severely retarded. Fatality is low, and many individuals survive well into adulthood. In most cases, the deletion appears for the first time in the affected individual (the parents being normal).

Down Syndrome (Trisomy 21) A condition caused by an extra chromosome 21. Individuals with Down Syndrome are usually of short stature, with a broad skull, round face, epicanthic fold, and mental retardation. About 1 in 6 children with this condition die within the first year, usually of congenital heart defects. The frequency of leukemia is 10-14 times higher in these individuals than in the population at large. The average life expectancy is about 30 years. Down Syndrome occurs with a frequency of 1 in 700 births, and is directly related to maternal age. 40% of Down Syndrome patients are born to women over 40, although they produce only about 4%0 of all births.

Edwards Syndrome (Trisomy 18) A condition associated with an extra for chromosome 18. Characterized by congenital malformations which affect many organ systems. Low set, faunlike ears with pointed pinnae are characteristic. The hands are usually tightly closed in fists, with the index finger folded to overlap the third digit, and the fifth overlapping the fourth digit. Cardiac malformations include septal defects. Average survival is 2-3 months for males and 10 months for females.

Patau Syndrome (Trisomy 13) A condition caused by an extra for chromosome 13. The phenotype is somewhat variable, but includes mental retardation, cleft lip and/or palate, deafness, cardiac malformations, and posterior protrusions of the heel. Most children with this condition die within the first three months of life. This condition, caused by chromosome nondisjunction, occurs with a frequency of 1 in n 5000 births.

Turner Syndrome (45,X) The first chromosomal anomaly involving the sex chromosomes to be described. Affected individuals are females, having only one X chromosome. The phenotypic characters include small stature, swelling of the hands and feet as infants, and an excess of skin at the nape of the neck. Overall, the symptoms are not usually severe, and the survival rate is not different from the normal population. This condition, arising from chromosomal nondisjunction, occurs with a frequency of 1 in 3000 female births.

Wilms Tumor A chromosomal abnormality associated with a deletion within the short arm of chromosome 11. This internal deletion results in aniridia (absence of the iris), mental retardation, microcephaly, and malignant tumors of the kidney. Tumor growth usually occurs in infants and young children, and leads to death.

Klinefelter Syndrome (47,XXY) A chromosomal abnormality caused by the presence of two X chromosomes in addition to one Y chromosome. Individuals with this condition are phenotypically male, but often show tendency toward female secondary sexual characteristics, such as enlarged breasts. Diagnosis is usually at puberty. Individuals are sterile. Intellectual development is in the normal range, and survival is also within the normal range. Chromosomal nondisjunction is the cause of this condition, which occurs with a frequency of 2 in 1000 male births. In a small percentage of cases, more complex karyotypes such as 48, XXXY are seen. In general, higher numbers of-sex chromosomes are associated with the development of mental retardation.

XXX Syndrome. The 47,XXX female karyotpye was first described in 1959 and has been shown to occur in about 1 in 1200 births. In most cases, the phenotype is completely normal, including fertility. There is a slight disturbance in intellectual development in two-thirds of the cases, but otherwise, the addition of an extra chromosome has little or no effect on the individual. Normality is attributed to the inactivation of two X chromosomes (as Barr bodies) in somatic cells.

XYY Syndrome. This chromosomal condition was first described in 1961, and subsequent population surveys revealed that 7 XYY males were detected in a group of 197 mentally retarded males institutionalized for violent or criminal tendencies. On this basis, it was suggested that the presence of an extra Y chromosome might predispose individuals toward aggressive behavior. XYY is a common condition, occurring with a frequency of 1 in 1000 male births, and only a small percentage of these individuals (about 3.5%) are ever institutionalized. A wide range of phenotypes is present in this condition, but only two, tallness and subnormal intelligence, are constant.

Practice Karyotypes

Carefully cut out the individual chromosomes from the photographed metaphase spread. Using the sample karyotype in Figure 7 as a guide, arrange each chromosome on the blank karyotype form. Do not fasten the chromosomes to the karyotype form or throw away any paper scraps until you have identified each chromosome.

The A group consists of six chromosomes. The number 1 pair is the longest and has the centromere in the middle. Number 2 is the second longest and has the centromere slightly off centre Number 3 is the third longest with the centromere in the middle. The B group consists of four long chromosomes with the centromeres very close to one end. If the preparation is not banded, there is no way to determine which two belong to pair 4 and which is pair 5. The C group consists of 14 medium-sized chromosomes with the centromeres slightly off-center. The X chromosome also falls in this group. Therefore, a male will have 15 C-type chromosomes and a female will have 16. Frequently, the X chromosome is the largest in the C group. The D group consist of six chromosomes slightly smaller than the C's with the centromeres very near one end. The E group resembles the C group but the chromosomes are much smaller. The F group chromosomes are very small with the centromeres in the middle. The G group includes the four smallest chromosomes with-the centromeres so close to the ends that it is difficult to see any short arms at all. The Y chromosome falls into the G group. Therefore, a male will have five G-type chromosomes and a female four. The Y chromosome can often be separated from the other members of the G-group by its parallel long arms if unbanded.

When you have completed the normal karyotype attempt one of the abnormal chromosome complements. First, count the chromosomes. The normal number is 46. If there are more than 46 chromosomes you know there is extra chromosomal material. If there are less than 46, there are two possibilities: either a chromosome has been lost or two chromosomes have joined. Now, cut out the chromosomes and prepare the karyotype by matching the chromosome pairs. Remember, a female has two X chromosomes and a male has one X and one Y chromosome. Compare your abnormal karyotype with a normal one to identify the type of abnormality.