Islamic University of Gaza. Human Karyotype. Training Course Manual. Prepared by: Mazen Ali Abo Zayed

Transcription

1 Islamic University of Gaza Human Karyotype Training Course Manual Prepared by: Mazen Ali Abo Zayed 1

2 Table of contents Title Page No. 1. Objectives 3 2. Introduction to Karyotyping 3 3. Materials and Methods Materials required Methods Peripheral blood media preparation Blood preparation Karyotyping procedure Staining Procedure Karyotype analysis Classification of Chromosomes for Karyotyping Examples of chromosomal aberrations Human disorders due to chromosome alterations in autosomes Nondisjunction of the sex chromosomes 13 2

3 Human Karyotype 1. Objectives: There are several genetic disorders that involve entire chromosomes. The objectives of this lab will be: 1. To demonstrate a microtechnique for reliable chromosomal analysis of leucocytes obtained from peripheral blood. 2. To prepare a karyotype from the chromosomes of human male or female. 3. To use the karyotyping techniques for diagnosing a chromosomal disorder. 2. Introduction to Karyotyping: Chromosomal aberrations are abnormalities in the number or microscopically observable structure of chromosomes. The number of chromosomes in human cells is 46 with 22 autosomal pairs (one of each type contributed by the mother and one of each type from the father) and 2 sex chromosomes - 2 X chromosomes for females (one from father and one from mother) or an X and a Y chromosome for males (the X from the mother and the Y from the father). The chromosomes visible only at the metaphase stage of mitosis, 22 homologous pairs of autosomes and two sex chromosomes. Each chromosome has a characteristic size and shape in the normal cell. During most of the cell cycle, interphase, the chromosomes are somewhat less condensed and are not visible as individual objects under the light microscope. Mitosis, or nucleus division, is the first part of M-phase and in consists of four stages (prophase, metaphase, anaphase and telophase). However during cell division, mitosis, the chromosomes become highly condensed and are then visible as dark distinct bodies within the nuclei of cells. The chromosomes are most easily seen and identified at the metaphase stage of cell division. 3

4 Cell Cycle = Interphase + M phase Interphase = G1phase: initial growth cycle, S phase: DNA synthesis (replication), G2 phase: second growth stage. M phase = Mitosis + cytokinesis The banding of chromosomes by using dyes was discovered in the late 1960's and before that cytogeneticists depended on chromosome length and position of a constriction to identify the individual chromosomes. The band width and the order of bands is characteristic of a particular chromosome - a trained cytogeneticist can identify each chromosome (1,2,3...22, X and Y) by observing its banding pattern under the microscope. chromosomes are arranged and numbered by size, from largest to smallest. This arrangement helps scientists quickly identify chromosomal alterations that may result in a genetic disorder. Identifying chromosomes has become easier in recent years by using certain staining techniques. One of the most common staining techniques involves giemsa stain, which gives the chromosomes a banded appearance (hence giemsa banding or G-banding) G-banding is the treatment of chromosomes in the metaphase stage with trypsin (to partially digest the protein) and stain them with Giemsa. Each homologous chromosome pair has a unique pattern of G-bands, enabling recognition of particular chromosomes. Karyotyping is the process by which doctors and geneticists take pictures of the chromosomes while the cell are undergoing mitosis. The picture is then enlarged. The picture of the chromosomes are then cut up so that each chromosome is removed. The chromosomes are matched up and attached to a paper according to size. The chromosomes pairs are numbered from largest to smallest. There are 22 pairs of chromosomes which match up exactly. Then the sex chromosomes are paired, in the female (XX) the chromosomes match and in the male (XY) the chromosomes do not match. This technique can be used to assess the normalcy of an individual s chromosomes and to assay for various genetic diseases such as Down s syndrome and 4

5 Klinefelter s syndrome. It is estimated that one in 156 live births have some kind of chromosomal abnormality. A chromosome is divided by its centromer into short arm (p) and long arm (q). chromosomes can be classified by the position of their centromer: - Metacentic: If its two arms are equal in length. - Submetacentric: If arms' lengths are unequal. - Acrocentric: If the p arm is so short that is hard to observe, but still present The photograph is enlarged and cut up into individual chromosomes. The homologous chromosomes can be distinguished by length and by the position of the centromer so the chromosomes can be arranged in 7 groups (A, B, C, D, E, F, G). Karyotypes are arranged with the short arm of the chromosome on top, and the long arm on the bottom. In addition, the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms. For example, Cri du chat syndrome involves a deletion on the short arm of chromosome 5. It is written as 46,XX,5p-. The critical region for this syndrome is deletion of 15.2, which is written as 46,XX,del(5)(p15.2). Peripheral Blood Karyotyping Medium With Phytohemagglutinin (PHA) is intended for use in short-term cultivation of peripheral blood lymphocytes for chromosome evaluation. It is based on RPMI-1640 basal medium supplemented with L-Glutamine, fetal bovine serum and antibiotics (penicillin and streptomycin). Karyotyping Medium is supplied as frozen medium, which is ready for use after thawing and phytohaemagglutinin supplementation. The blood cell karyotyping method was developed to provide information about chromosomal abnormalities. Lymphocyte cells do not normally undergo subsequent cell divisions. In the presence of a mitogen (PHA), lymphocytes are stimulated to enter into mitosis by DNA replication. After hours, a mitotic inhibitor (colcemid) is added to the culture to stop mitosis in the metaphase stage. After treatment by hypotonic solution, fixation and staining, chromosomes can be microscopically observed and evaluated for abnormalities. 5

6 Alterations in chromosome number: Nondisjunction occurs when either homologues fail to separate during anaphase I of meiosis, or sister chromatids fail to separate during anaphase II. The result is that one gamete has 2 copies of one chromosome and the other has no copy of that chromosome (The other chromosomes are distributed normally). If either of these gametes unites with another during fertilization, the result is aneuploidy (abnormal chromosome number) A trisomic cell has one extra chromosome (2n +1) = example: trisomy 21 ( Down syndrome). A monosomic cell has one missing chromosome (2n - 1) = usually lethal except for one known in humans: Turner's syndrome (monosomy XO). The frequency of nondisjunction is quite high in humans, but the results are usually so devastating to the growing zygote that miscarriage occurs very early in the pregnancy. If the individual survives, he or she usually has a set of symptoms - a syndrome - caused by the abnormal dose of each gene product from that chromosome. 6

7 Translocation is the result of chromosomal breakage but the broken segment transfers itself to a broken segment of another chromosome. There are both balanced and unbalanced translocations. Deletion - Deletion occurs when a chromosome breaks and a portion of the chromosome is lost. Inversion - a section of the chromosome is inverted (reversed) on the same chromosome. 3. Materials and methods: 3.1 Materials required: Peripheral human blood Heparin sodium injection 25000units/vial Karyotype medium RPMI-1640 Fetal Bovine Serum Penicillin Streptomycin U/ml penicillin and µg/ml streptomycin Phytohemagglutinin-M (PHA-M) L-glutamin 200 mmol/l Colcemid solution 10µg/ml Hypotonic solution (0.075M KCl) 3X Absolute Methanol +1X Glacial Acetic Acid Giemsa stain Hanks buffer Trypsine Slides and microscope 3.2 Methods: Peripheral blood media preparation: Blood culture media; 500 ml RPMI 1640 with 100ml fetal bovine serum, 6.5ml penicillin streptomycin and 7ml glutamine. Dispense 10ml aliquots into sterile tube and add 2% (0.2ml) PHA to each tube. Store at 4 C for along as 2 weeks. 7

8 Blood preparation:. 1- Wash 10ml syringe with Heparin solution and collect blood aseptically to the syringe (0.5ml for each tube). 2- Remove the blood to sterile vacuette tubes or to sterile, Heparin prewashed tube. 3- Store at 4 o C until required (upto 7 hours). 4- Before use, invert the tube with blood several times Karyotyping procedure: 1- Inoculate 0.5ml of heparinized whole blood into tube with 10ml of karyotyping medium 2- Incubate the tubes in incubator with 5% CO 2 at 37ºC for total of hours. 3 -After total of 69 hours from seeding add 100μl of Colcemid Solution to each culture tubes. 4 - Incubate the tubes at 37 o C for an additional minutes. 5 - Spin at 500g (1500RPM) for 7 minutes. 6 - Remove the supernatant and re-suspend the cells in 5ml of hypotonic 0.075M KCl pre wormed to 37ºC. 7- Incubate at 37 o C for 15 minutes. 8- Add drop-by- drop (with vortexing) 1ml fresh ice cold fixative. 9- Spin at 500g (1500RPM) for 7 minutes. 10- Remove the supernatant, agitate the cellular sediment and add drop-bydrop (with continous vortexing), 5ml of fresh, ice-cold fixative. 11- Leave at 4ºC for 20 minutes. 12- Repeat steps 9 and 10,until the supernatant is clear. 13- Spin at 500g (1500RPM) for 7 minutes. 14- Re-suspend the cell pellet with a 1.5ml of fresh fixative. 15- Drop 4-5 drops, from a high of approximately 50 cm onto a clean slide and blow carefully on the drops for spreading them on the slide. 16- Heat the slides to 55ºC for overnight. 8

9 Staining Procedure: The staining procedure should be done in a 37ºC water bath. Put the slides in a staining rack (e.g. coplin staining jar) and treat as follows: 1-2 minutes in 50ml Hanks solution minutes in: 47.5ml Hanks solution + 2.5ml Trypsin-EDTA solution. 3- Wash in: 40ml Hanks solution +10ml Fetal bovine serum. 4- Wash in: 50ml Hanks solution minutes in: 47ml Buffer solution ph ml Giemsa stain solution. 6- Wash several times with 50ml Buffer solution ph Air dry the chromosome slides. 8- Check for chromosome spreads in a phase contrast lab microscope Karyotype analysis: 1- Obtain a set of chromosomes. 2- Match the chromosomes with their homologous mate. One chromosome of each pair is numbered, as you match your chromosomes number the homologous pairs. You need to be very systematic.the number one chromosome is the largest. Its corresponding mate should be of the same size, with the same banding pattern, and have the same centromere location. 3- Determine the karyotype abnormality using the Chromosome Analysis Key that is below. Keep in mind there might be some slight variations. The key below will help guide you. 4- Research your abnormality using the resources available. A photograph of a real karyotype for the disorder is in the computer database, for example BandView is Applied Spectral Imaging's fullyautomated image acquisition and analysis system for Karyotyping (High resolution images with 12-bit digital CCD-camera (1280x1024 pixels). 5- Simple key for diagnosing chromosome abnormalities : 1A 46 chromosomes in karyotype... GO TO STATEMENT 3 1B Not 46 chromosomes in karyotype... GO TO STATEMENT 2 9

10 2A 47 chromosomes in karyotype (3 of one chromosome)... Trisomy condition 2B One chromosome missing from pair... Monosomy condition 3A All chromosomes are matched with its homologue without obvious missing pieces or additions Possible Normal individual 3B Homologous pairs are not identical in size... GO TO STATEMENT 4 4A There are additions pieces attached to a chromosome... Translocation 4B There are missing pieces in a chromosome... Deletion Classification of Chromosomes for Karyotyping: Chromosomes are arranged into seven groups based on size and centromere location. The centromeres can be found in the middle of the chromosome (median), near one end (acrocentric), or in between these first two (submedian) Group A: chromosomes 1-3 are largest with median centromere. Group B: chromosomes 4-5 are large with submedian centromere Group C: chromosomes 6-12 are medium sized with submedian centromere Group D: chromosomes are medium sized with acrocentric centromere 10

11 Group E: chromosomes are short with median or submedian centromere Group F: chromosomes are short with median centromere Group G: chromosomes are very short with acrocentric centromere. Chromosome X is similar to group C. Chromosome Y is similar to group G 4. Examples of chromosomal aberrations: 4.1. Human disorders due to chromosome alterations in autosomes (Chromosomes 1-22). There only 3 trisomies that result in a baby that can survive for a time after birth; the others are too devastating and the baby usually dies in utero. Trisomy 13, XX Patau Syndrome. The karyotype here demonstrates trisomy 13 (47, XX, +13). It is rare for fetuses with this condition to go to term, so it occurs in only 1 in 15,000 live births. It is rare for babies to survive for very long if liveborn because of the multitude of anomalies that are usually present. Forty five percent die within the first month, 90% by six months and less than 5% reach 3 years. There is severely abnormal cerebral functions and virtually always leads to death in early infancy. This baby has very pronouced clefts of the lip and palate, broad nose, small cranium, polydactyl (An extra finger), deafness, and nonfunctional eyes. Heart defects and severe mental retardation are also part of the clinical picture. 11

12 Trisomy 18 (Edwards Syndrome): Edwards Syndrome. This karyotype demonstrates trisomy 18 (47, XY, +18). It is uncommon for fetuses with this condition to survive, so the incidence is only 1 in 8000 live births. Thirty percent of these children die within the first month and only 10% survive one year. It is rare for babies to survive for very long if liveborn because of the multitude of anomalies that are usually present. There is severe mental retardation and a highly characteristic pattern of malformations such as elongated skull, a very narrow pelvis, rocker bottom feet and a grasping of the two central fingers by the thurm and little finger. In addition, the ears are often low set and the mouth and teeth are small. Nearly all babies born with this condition die in early infancy. Trisomy 21 (Down syndrome) This is an example of trisomy 21 (47, XY, +21) also known as Down syndrome. Note the extra chromosome 21. Additions or deletions of genetic material are generally lethal in utero. Trisomy 21 is an example of one form of addition in which the fetus may occasionally survive to term and beyond. Of those born with Down syndrome, 1/6 die within the first year and the average life span is 16.2 years. The non-dysjunctional event in meiosis that produces this anomaly increases in incidence with increasing maternal age. Trisomy 21, one of the most common causes of mental retardation. The child can have an IQ between An average person has an IQ between This results in a number of characteristic features, such as short stature, broad 12

13 hands, stubby fingers and toes, a wide rounded face, a large protruding tongue that makes speech difficult. Individuals with this syndrome have a high incidence of respiratory infections, heart defects, and leukemia. The average risk of having a child with trisomy 21 is 1/750 live births. Mothers in their early twenties have a risk of 1/1,500 and women over 35 have a risk factor of 1/70, which jumps top 1/25 for women 45 and over. Trisomy 16 with Monosomy X (Never survive): Here is an example of trisomy 16. This is the most common chromosomal abnormality, but the fetuses NEVER survive past the first trimester. Many first trimester fetuses are lost in this fashion (many are "silent" abortions). Note in this case that a sex chromosome is missing as well. Intrauterine demise is nature's way of eliminating abnormal karyotypes Nondisjunction of the sex chromosomes (X or Y chromosome): Can be fatal, but many people have these karyotypes and are just fine! Klinefelter syndrome: 47, XXY males. Male sex organs; unusually small testes, sterile. Breast enlargement and other feminine body characteristics. Normal intelligence. 13

14 This is Klinefelter's syndrome with a 47, XXY karyotype. A non-dysjunctional event in meiosis left two X chromosomes in an ovum. This particular anomaly is relatively common (about 1 in 500 males), with affected persons being relatively normal. Characteristics associated with this condition are tall stature and sterility. XXXXY Variation of Kleinfelter's Syndrome 49,XXXXY. This karyotype shows a variant of Klinefelter's syndrome. Individuals with this syndrome are male, typically with the karyotype 47,XXY. They exhibit a characteristic phenotype including tall stature, infertility, gynecomastia and hypogonadism. Aneuploidy above one extra chromosome is usually fatal but because of X-inactivation, which "turns off" all but one X chromosome per cell, the effects of 3 extra chromosomes are reduced. 14

15 47, XYY males (Jacobs Syndrome): Individuals are somewhat taller than average and often have below normal intelligence. At one time (~1970s), it was thought that these men were likely to be criminally aggressive, but this hypothesis has been disproven over time. Jacobs Syndrome. A chromosome aberration which is caused by nondisjunction of the Y chromosome during the second phase of meiosis giving a 47 XYY karyotype. Occurence is 1/1000 live male births. Men with this karyotype are tall and have low mental ability. Trisomy X: 47, XXX females: 1:1000 live births - healthy and fertile - usually cannot be distinguished from normal female except by karyotype 15

16 Monosomy X (Turner's syndrome): This is monosomy X (Turner's syndrome, with karyotype 45, XO). This can occur in about 1 per 2,700 births. It is not linked to maternal age. Women with Turner's syndrome can live relatively normal lives, though they are unable to bear children. The phenotype of this female includes short stature, short broad neck, and a broad chest. Intelligence does not seem to be affected. (98% of these fetuses die before birth) 16