Karyotyping: Part 1 and 2 PDF

Karyotyping Part 1 Mark John R. Ravina, RMT  A karyotype is the number and Karyotype appearance of chromosomes in the nucleus of a eukaryotic cell. Analyzes: ❑Sex chromosome content ❑Presence or absence of...

Karyotyping Part 1 Mark John R. Ravina, RMT  A karyotype is the number and Karyotype appearance of chromosomes in the nucleus of a eukaryotic cell. Analyzes: ❑Sex chromosome content ❑Presence or absence of individual chromosomes ❑Nature or extent of chromosomal aberrations The term is also used for the complete set of chromosomes in a species, or an individual organism.  Karyotypes may be performed by taking (1) a blood, bone marrow, or skin sample from an adult; or (2) a sample of amniotic fluid (which contains stray cells) or extra-embryonic cells (from the chorionic villi) from an unborn child. Once the cells have been obtained for a karyotype, they are mixed with plant-derived chemicals called lectins that stimulate mitosis. Karyotyping After the required quantity of cells has been acquired, the cells are treated with a drug called colchicine to stop the mitotic Analysis process at metaphase. Colchicine arrests the action of spindle fiber microtubules; hence ceasing the mitotic process. The cells are placed in a hypotonic solution that causesthem to take on water, thereby increasing their overall size. This provides room for chromosomesto be spread out. The cells are fixed to a microscope slide, stained and then photographed.  After they are photographed, the photo is enlarged and the chromosomes are cut out and matched up as pairs. Chromosomes may be distinguished from one another based upon several key characteristics: i. the length of the arms of the chromosome Karyotyping ii. shape iii. general appearance of the chromosome Analysis Karyotype analysis provides a mechanism to determine if non- disjunctionaldiseases, such as Down syndrome (Trisomy 21), are present in the fetus. It also provides a look at the chromosomes to see if there are any missing segments (deletions) or translocations that may have occurred during crossing-over. Normal Cell Cancer Cell  Variations in Chromosome Number Polyploidy – a chromosome number that is a multiple of the normal haploid set. Aneuploidy – a chromosomal number that varies by something less than a set Monosomy – having only one member of a homologous pair. Trisomy – having three copies of a single chromosome. Aneuploidy is a Major Cause of Reproductive Failure ❑ It is estimated that humans have a rate of aneuploidy 10x higher than other mammals, including primates. ❑ 1 in 2 conceptions are aneuploid ❑ 35%-70% of early embryonic deaths and spontaneous abortions are caused by aneuploidy ❑ 1 in 170 live births are at least partially aneuploid ❑ 5%-7% of early childhood deathstare related to aneuploidy.  Polyploidy ❑Caused by: i. Errors in meiosis ii. Events after fertilization iii. Errors in mitosis Triploidy Tetraploidy ❑ Three sets of chromosome ❑ Four sets of chromosome ❑ Most common form of ❑ 5% of all spontaneous polyploidy abortions ❑ 15%-18% of all spontaneous ❑ Extremely uncommon in live abortions births ❑ Approximately 75% have two ❑ May result from failure of sets of paternal chromosomes cytokinesis in the first ❑ Probably due to polyspermy mitotic division ❑ 1% conceptions are triploid ❑ Life threatening but 99% die before birth  Most Common Cause of Nondisjuction is the failure of homologs or sister chromatids Aneuploidy in chromosomes to separate in meiosis or mitosis ✓ Produces abnormal gametes Nondisjunction in Meiosis ✓ Phenotypic effects of aneuploidy Autosomal Monosomy Autosomal Trisomy i. Lethal condition i. Most are lethal ii. Aneuploidy during gamete formation ii. 50% of cases of chromosomal abnormalities produces equal numbers of monosomic and that cause fetal death are autosomal trisomies trisomic gametes and embryos. iii. Varies by chromosome: iii. Rarely seen in spontaneous abortions and live ✓ Trisomy 13: Patau Syndrome (47,+13) births ✓ Trisomy 18: Edwards Syndrome (47, +18) iv. Majority are lost early in development ✓ Trisomy 21: Down Syndrome (47, +21) Trisomy 18: Edwards Syndrome (47, +18) i. ii. 1/10,000 births Average survival time: 2-4 months iii. Affected infants small at birth grow slowly and are mentally retarded iv. Malformation of heart, hands and feet v. For unknown reasons 80% of all trisomy 18 are female vi. Advanced maternal age is a risk factor Trisomy 13: Patau Syndrome i. 1/5,000births (47, +13) ii. Lethal: mean survival time is at least 1 month iii. Facial malformations, eye defects, extra fingers or toes, and large protruding heels iv. Severe malformations of brain, nervous system, and heart v. Parental ageonly known risk factor Trisomy 21: Down Syndrome (47, +21) i. First chromosomal abnormality discovered in humans (1959) ii. 1/700 live births iii. Leading cause of mental retardation and heart defects in US iv. Wide flat skulls, skin folds in the corner of the eyes, spots on the irises, and thick-furrowed tongues. v. 40% congenital heart defects.  i. Advanced maternal age Risk for Autosomal Trisomy ii. Risk increases rapidly after 35 years of age  Klinefelter’s Syndrome (47, XXY) 1/1000 Featuresdo not developuntil after puberty Affectedindividuals aremalewith low fertility and may have mental dysfunction 60% due to maternal nondisjunction Other forms XXYY, XXXYand XXXXY Turner Syndrome (45, X) 1/5000births Females; short, wide chest; rudimentary ovaries; and abnormal sexual development Puffiness of hands and feet Abnormalities of the aorta No mental dysfunction Single Xchromosome; two X chromosomes are required for normal female sexual development Complete absenceof anXchromosome is lethal- (So,no Y monosomies) Jacob Syndrome (47, XYY) 1/1000 births Above average in height No established link with possible antisocial behavior Thank You FOR LISTENING! KARYOTYPING P A R T 2 M a r k J o h n R. R a v i n a , R M T ❑ The study of karyotypes is made possible by staining. ❑ Giemsa is applied after cells have been arrested during cell division by a solution of colchicine. ❑ For humans, WBC are used most frequently because they are easily induced to divide and grow in tissue culture. ❑ Sometimes observations may be made on non-dividing (interphase cells) ❑ The sex of an unborn fetus can be determined by observation of interphase cells (amniocentesis and Barr body) ❑ A normal human female has only one Barr body per somatic cell, while a normal human male has none. ❑ A Barr body is the inactive X chromosome in a female somatic cell Cells Used For Chromosomal Analysis i. Any cell with a nucleus ii. Lymphocytes iii. Skin cells iv. Tumor cells v. Amniotic cells vi. Chorionic villi vii. Rare fetal cells from maternal blood  ❑ 5 mL of blood is removed from the patient. ❑ If a fetus is being karyotyped, amniotic fluid is removed from the amniotic sac which surrounds the fetus during development. ❑ This is done with the aid of a large syringe and ultrasound picturing. ❑ There are cells which have come off the fetus in this fluid. The WBC are removed from the blood or the living cells are removed from the amniotic fluid. ❑ These cells are then cultured in a medium in which they undergo mitosis ❑ Mitosis is stopped at metaphase using chemicals. KARYOTYPING ❑ The cells are then placed onto a slide and spread out. They are viewed under a microscope which is specially adapted with a camera to take a picture of the chromosomes from one of the cells. PROCEDURE ❑ Once the picture is taken and enlarged, the chromosomes are cut out and arranged in pairs according to size and location of the centromere  Group A Chromosomes 1-3 are largest with median centromere Chromosomes are arranged into seven groups based on sizeand centromere Group B Chromosome 4-5 are large with submedian location. The centromeres can be found in centromere the middle of the chromosome (median), Group C Chromosomes 6-12 are medium sized with near one end (acrocentric), or in between submedian centromere these firsttwo(submedian) Group D Chromosomes 13-15 are medium sized with acrocentric centromere Group E Chromosomes 16-18 are short with median or submedian centromere Group F Chromosome 19-20 are short with median centromere Group G Chromosomes 21-22 are very short with acrocentric centromere Chromosome X Similar to Group C Chromosome Y Similar to Group G Chromosome Painting Using Fluorescent Dyes ❑DNA sequences attached to fluorescent dyes ❑The sequences attach to the chromosome and “paint” specific regions ❑Using several different DNA sequences and fluorescent dyes produces a unique pattern for each of the 24 types of human chromosomes  6 Different i. ii. Differences in absolute sizes of chromosomes. Differences in relative size of chromosomes. Characteristics of iii. iv. Differences in the position of centromeres Differences in basic number of chromosomes Karyotypes are Usually v. Differences in number and position of satellites vi. Differences in degree and distribution of Observed and Compared heterochromatic regions.  PROCEDURE: 1. De-stain slides for 10 minutes in 95% ethanol 2. Place slide in PBS ( phosphate Types of Banding Buffer Solution) and incubate for 10 MINUTES AT 56OC 3. Treat slide with 0.22 mL of 25 % ❑ Cytogenetics employs several techniques to visualized trypsin, 2.5 mL of methanol and different aspects of chromosomes: 0.22 mL of stock Giemsa and 6.5 i. G-banding mL 0f PBS with a pH of 7.4. ii. R-banding iii. C-banding 4. Flood slide with stain solution for iv. Q-banding 15 minutes. Rinse with water and v. T-banding air dry. 5. View slide under bright field, oil immersion microscope.  G-banding R-banding C-banding i. G-banding is obtained with Giemsa stain i. R-banding is the reversed of G-banding i. Giemsa binds to consecutive following digestion of chromosomes with o The dark region are euchromatic heterochromatin, so it stains centromeres. trypsin. (guanine-cytosine rich regions) ii. It yields a series of lightly and darkly o The bright/light regions are stained bands heterochromatic (thymine-adenine Q-banding o The dark regions tend to be rich regions) i. Q-banding is a fluorescent pattern heterochromatic, late-replicating ii. A reverse Giemsa chromosome banding obtained using quinacrine for staining. The and AT rich. method that produces bands pattern of bands is very similar to that o The light regions tend to be complementary to G-bands; induced by seen in G-banding. euchromatic, early-replicating and treatmentwith high temperature, low pH, or GC rich acridine orange staining; often used iii. This method will normally produce 300- together with G-banding on human T-banding 400 bands in a normal, human genome karyotype to determine whether there are deletions. i. Visualize telomeres Silverstaining:Silvernitrate stains the nucleolar organization region-associated protein. This yields a dark region where the silver is deposited, denoting the activity of rRNA genes within the NOR. 单击此处添加标题 SPECTRAL KARYOTYPING ❑ Spectral karyotyping is a molecular cytogenetic technique used to simultaneously visualize all the pairs of chromosomes in an organism in different colors. ❑ Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. ❑ Because there are a limited number of spectrally- distinct fluorophores, a combinatorial labeling method isused to generate many different colors. ❑ Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope. ❑ Image processing software then assigns a pseudo color to each spectrally different combination, allowing the visualization of the individually colored chromosomes. ❑ This technique is used to identify structural chromosome aberrations in cancer cells and other dis ease conditions when Giemsa banding or other techniques are not accurate enough. 单击此处添加标题 DIGITAL KARYOTYPING Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale. Shortsequences of DNA from specific loci all over the genome are isolated and enumerated. Thismethod isalso known as virtual karyotyping Variation is often found: i. Between thesexes ii. Between the germ-line and soma iii. Between members of a population iv. Geographical variation betweenraces v. Mosaics or otherwise abnormal individuals. ❑ The normal human karyotypes contain22 pairs of autosomal chromosomes and one pair of sex chromosomes. ❑ Normal karyotypes for females contain two X chromosomes and are denoted46,XX; males have both an X and a Y chromosome denoted 46, XY. ❑ Any variation from the standard karyotype may lead to developmental abnormalities. CHANGES DURING DEVELOPMENT Some organisms go in for large-scale elimination of heterochromatin, or other kinds of visible adjustment to the karyotype. Chromosome Chromatin X-inactivation Elimination Diminution o Chromosome abnormalities can be numerical, as in the presence of extra or missingchromosomes,or structural, as in derivative chromosome, translocations, inversions, large- scale deletions or duplications. o Numerical abnormalities, also known as aneuploidy, often occur as a result of nondisjunction during meiosis in the formation of a gamete; o Trisomies, in which three copies of a chromosome a re present instead of the usual two, are common numerical abnormalities. o Structural abnormalities often arise from errors in homologous recombination.  Ploidy isthe number of complete sets of chromosomes in a cell. Polyploidy Haplo-diploidy Endopolyploidy Aneuploidy where there are more where one sexisdiploid, A process by which It isthe condition in than two setsof and the other haploid. Itis chromosomes replicate which the chromosome homologous a common arrangement in without the division of number inthe cells isnot chromosomes in the cells, the Hymenoptera, and in the cell nucleus,resulting the typical number for occurs mainly in plants. some other groups. in a polyploid nucleus. the species. Also called endomitosis. Some disorders arise from loss of just piece of one chromosome ❑ Cri du chat (cry of the cat), from a truncated short arm on chromosome 5. The name comes from the babies' distinctive cry, caused by abnormal formation of the larynx. ❑ 1p36 Deletion syndrome, from the loss of part of the short arm of chromosome1. ❑ Angelman syndrome –50%of cases have a segment of the long arm of chromosome 15 missing;a deletion of the maternal genes, example of imprintingdisorder. ❑ Prader-Willi syndrome –50%of cases have a segment of the long arm of chromosome 15 missing;a deletion of the paternal genes, example of imprintingdisorder. ❑ Chromosomal abnormalities can also occur in cancerous cells of an otherwise genetically normal individual; ❑ One well-documented exampleis the Philadelphia chromosome, a translocation mutation commonly associated with chronic myelogenous leukemia and lessoften with acute lymphoblastic leukemia. THANK YOU For listening!