Cytogenetics: Karyotyping and Chromosomal Aberrations Lecture Notes PDF

Michael Cummings Karyotyping and Chromosomal Abberations Prof. Dr. Kalaivani Nadarajah David Reisman University of South Carolina  Karyotypes and Chromosome Aberrations The Human Chromosome Set The number and appearance of chromosomes in the nucleus of an...

Michael Cummings Karyotyping and Chromosomal Abberations Prof. Dr. Kalaivani Nadarajah David Reisman University of South Carolina  Karyotypes and Chromosome Aberrations The Human Chromosome Set The number and appearance of chromosomes in the nucleus of an organism is an important characteristic Chromosome analysis is a powerful and useful technique in human genetics  Chromosome Number Chromosome number in selected organisms Loading…  Chromosome Shape As chromosomes condense and become visible during cell division, certain structural features can be recognized Centromere A region of a chromosome to which microtubule fibers attach during cell division The location of a centromere gives a chromosome its characteristic shape  Centromere Location Metacentric A chromosome that has a centrally placed centromere Submetacentric Loading… A chromosome whose centromere is placed closer to one end than the other Acrocentric A chromosome whose centromere is placed very close to, but not at, one end  Human Chromosomes Replicated chromosomes at metaphase consist of sister chromatids joined by a single centromere Fig. 6-1, p. 122  Metaphase Chromosomes Chromosomes are identified by size, centromere location, banding pattern  Metacentric Submetacentric Acrocentric Short Satellite arm (p) p Centromere p Stalk Long q q arm (q) 3 17 21  Types of Chromosomes Sex chromosomes In humans, the X and Y chromosomes that are involved in sex determination. These have different sizes and shapes Autosomes Chromosomes other than the sex chromosomes In humans, chromosomes 1 to 22 are autosomes  A Set of Human Chromosomes Human chromosomes are analyzed by construction of karyotypes Karyotype A complete set of chromosomes from a cell that has been photographed during cell division at the metaphase stage and arranged in a standard sequence A Human Karyotype Loading… Karyogram: Chromosome Banding Patterns  System of Naming Chromosome Bands Allows any region to be identified by a descriptive address (chromosome number, arm, region, and band)  Add a few Add phytohemagglutinin drops of blood. to stimulate mitosis. Draw 10 to 20 ml of blood. Incubate at 37°C for 2 to 3 days. Transfer to tube Transfer Add Colcemid to containing fixative. cells to tube. culture for 1 to 2 hours to stop mitosis in metaphase. Centrifuge to concentrate cells. Add low-salt solution to eliminate red blood cells and swell lymphocytes. Drop cells onto microscope slide. Examine with Digitized microscope. chromosome Stain slide images processed with Giemsa. to make karyotype. Metaphase Chromosomes (a) Arranged Into a Karyotype (b)  Constructing and Analyzing Karyotypes Karyotype construction and analysis are used to identify chromosome abnormalities Different stains and dyes produce banding patterns specific to each chromosome Karyotypes reveal variations in chromosomal structure and number 1959: Discovery that Down syndrome is caused by an extra copy of chromosome 21 Chromosome banding and other techniques can identify small changes in chromosomal structure Information Obtained from a Karyotype Number of chromosomes Sex chromosome content Presence or absence of individual chromosomes Nature and extent of large structural abnormalities Four Common Chromosome Staining Procedures  Banding technique Appearance of chromosomes G-banding — Treat metaphase Darkly stained G bands. spreads with trypsin, an enzyme that digests part of chromosomal protein. Stain with Giemsa stain. Observe banding pattern with light microscope.  Banding technique Appearance of chromosomes Q-banding — Treat metaphase Bright fluorescent bands spreads with the chemical upon exposure to quinacrine mustard. Observe ultraviolet light; same as fluorescent banding pattern with a darkly stained G bands. special ultraviolet light microscope.  Banding technique Appearance of chromosomes R-banding — Heat metaphase Darkly stained R bands spreads at high temperatures to correspond to light bands achieve partial denaturation of DNA. in G-banded chromosomes. Stain with Giemsa stain. Observe Pattern is the reverse of G- with light microscope. banding.  Banding technique Appearance of chromosomes C-banding — Chemically treat metaphase spreads to extract DNA Darkly stained C band from the arms but not the centromeric region of the centromeric regions of chromosome corresponds chromosomes. Stain with Giemsa to region of constitutive stain and observe with light heterochromatin. microscope. Chromosomal Aberrations and Specific Syndromes  Chromosome Painting New techniques using fluorescent dyes generate unique patterns for each chromosome  Obtaining Cells for Chromosome Studies Any nucleus can be used to make karyotype Lymphocytes, skin cells, cells from biopsies, tumor cells Sampling cells before birth Amniocentesis Chorionic villus sampling (CVS)  Amniocentesis A method of sampling the fluid surrounding the developing fetus by inserting a hollow needle and withdrawing suspended fetal cells and fluid Used in diagnosing fetal genetic and developmental disorders Usually performed in the sixteenth week of pregnancy Amniocentesis  Removal of about 20 ml of amniotic fluid containing suspended cells that were sloughed off from the fetus A few biochemical Centrifugation analyses with some of the amniotic fluid Quick determination of fetal sex and analysis Fetal cells of purified DNA Growth for Biochemical analysis for several days the presence of alleles in culture that cause many different medium metabolic disorders Karyotype analysis (a)  Chorionic Villus Sampling (CVS) A method of sampling fetal chorionic cells by inserting a catheter through the vagina or abdominal wall into the uterus Used in diagnosing biochemical and cytogenetic Loading… defects in the embryo Usually performed in the eighth or ninth week of pregnancy Chorionic Villus Sampling  Chorionic villi Developing Ultrasound to monitor placenta procedure Developing Bladder fetus Uterus Chorion Catheter Amniotic cavity Rectum (a)  Exploring Genetics: Noninvasive Prenatal Diagnosis Methods are being investigated to isolate fetal cells that can pass into the mother’s bloodstream (placental cells, white blood cells, immature red blood cells) for genetic testing  Variations in Chromosome Number Changes in chromosome number or chromosome structure can cause genetic disorders Two major types of chromosomal changes can be detected in a karyotype A change in chromosomal number A change in chromosomal arrangement  Changes in Chromosome Number Polyploidy A chromosomal number that is a multiple (3n or 4n) of the normal haploid chromosomal number Aneuploidy A chromosomal number that is not an exact multiple of the haploid number  Polyploidy Changes the Number of Chromosome Sets Triploidy A chromosomal number that is three times the haploid number, having three copies of all autosomes and three sex chromosomes Tetraploidy A chromosomal number that is four times the haploid number, having four copies of all autosomes and four sex chromosomes A Triploid Karyotype A Triploid Infant  Keep In Mind Polyploidy results when there are more than two complete sets of chromosomes  Aneuploidy Involves the Gain or Loss of Individual Chromosomes Monosomy A condition in which one member of a chromosomal pair is missing; one less than the diploid number (2n – 1) Trisomy A condition in which one chromosome is present in three copies, and all others are diploid; one more than the diploid number (2n + 1)  Causes of Aneuploidy Nondisjunction The failure of homologous chromosomes to separate properly during meiosis Nondisjunction in Meiosis I Leads to Aneuploidy  Extra chromosome Nondisjunction (n + 1) Extra chromosome (n + 1) Missing chromosome (n − 1) Missing chromosome (n − 1) Meiosis I Meiosis II Gametes (a)  Nondisjunction Extra chromosom Normal division e (n + 1) Missing chromosome (n − 1) Normal (n) Normal (n) Meiosis I Meiosis II Gametes (b)  Effects of Monosomy and Trisomy Autosomal monosomy is a lethal condition Eliminated early in development (spontaneous abortion) Some autosomal trisomies are relatively common Most result in spontaneous abortion Three types can result in live births (13, 18, 21) Trisomies in Spontaneous Abortions  7.5 Survey of 4,088 spontaneous abortions 5 P e r 4 c e n 3 t a g 2 e o f 1 tr i s o 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 m i Chromosome number  Trisomy 13: Patau Syndrome (47,+13) A lethal condition Usually have polydactyly, eye defects, severe brain, nervous system & heart 1 in 10,000 births, most defects die within 1st month  Trisomy 18: Edwards Syndrome (47,+18) A lethal condition Survival only 2-4 mths 1 in 11,000 births 80% are females Very slow growth, Mental retardation, Heart malformations  Trisomy 21: Down Syndrome (47, +21) Occurs in 1/800 births Trisomy 21 is the only autosomal trisomy that allows survival into adulthood Mental retardation Characterized by epicanthic fold High risk of leukemia & Alzheimer’s (corner of eye) disease Few reach the age of 50 large furrowed tongues 40% have congenital  heart defects Trisomy 21: Down Syndrome (47,+21) Monosomy and trisomy involve the loss and gain of a single chromosome to a diploid genome ANIMATION: Nondisjunction To play movie you must be in Slide Show Mode PC Users: Please wait for content to load, then click to play Mac Users: CLICK HERE  What Are the Risks for Autosomal Trisomy? The causes of autosomal trisomy are unknown Factors that have been proposed include: Genetic predisposition Exposure to radiation Viral infection Abnormal hormone levels Maternal age is the leading risk factor for trisomy 94% of nondisjunctions occur in the mother  18 16 Risk for 14 Down syndrome T ri 12 s o 10 m y 8 2 1 6 / 1 4 , 0 2 0 0 b 15 20 25 30 35 40 >44 ir Maternal age (a) t  h 35 P e r c 30 e Maternal age and n trisomic conceptions t 25 a g 15 e o f c 10 li n i 5 c a ll y r 15 16 18 20 22 24 26 28 30 32 34 36 38 40 ≥42 e Maternal c (b) age  o Why is Maternal Age a Risk Factor? Meiosis is not completed until ovulation Intracellular events may increase risk of nondisjunction, resulting in aneuploidy Maternal selection Embryo-uterine interactions that normally abort abnormal embryos become less effective Age of the mother is the best known risk factor for trisomy  Aneuploidy of the Sex Chromosomes More common than autosomal aneuploidy Can involve both X and Y chromosomes A balance is needed for normal development At least one copy of the X chromosome is required for development Increasing numbers of X or Y chromosomes causes progressively greater disturbances in phenotype and behavior  Turner Syndrome (45,X) Monosomy of the X chromosome that results in female sterility. Other phenotypic characteristics but otherwise normal. Fig 6.20  Klinefelter Syndrome (47, XXY) Individuals (males) have some fertility problems but few additional symptoms Fig 6.22  XYY Syndrome (47,XYY) Affected individuals are usually taller than normal and some, but not all, have personality disorders Changes in the number of sex chromosomes have less impact than changes in autosomes  Structural Alterations Within Chromosomes Changes in the structure of chromosomes Deletion Duplication Translocation Inversion Structural Changes in Chromosomes  Deletions Deletions involve loss of chromosomal material Deletions of chromosomal segments are associated with several genetic disorders Cri du chat syndrome Prader-Willi syndrome Deletion in Chromosome 5 and cri du chat syndrome Associated with an array of malformations, the most characteristic of which is an infant cry that resembles a meowing cat due to defects in the larynx By comparing the region deleted with its associated phenotype, investigators have identified regions of the chromosome that carry genes involved in developing the larynx.  Translocations Translocation involves exchange of chromosome parts Often produces no overt phenotypic effects Can result in genetically imbalanced and aneuploid gametes Chromosomes can lose, gain, or rearrange segments  Robertsonian Translocation A translocation resulting in Down syndrome Robertsonian translocation makes Down syndrome a heritable genetic disease Potentially present in one in three offspring Robertsonian Translocation and Down Syndrome What Are Some Consequences of Aneuploidy? Aneuploidy is the leading cause of reproductive failure in humans Results in miscarriages and birth defects Aneuploidy also is associated with many cancers Chromosomal Abnormalities in Miscarriages  The frequency of aneuploidy changes dramatically over developmental time. Between 6 to 8 weeks and 20 weeks, about 35% of spontaneous abortions are aneuploid. Around 20 weeks, the frequency falls by an order of magnitude to about 4% stillbirths. Gametes Gestation (weeks) Sperm Eggs 0 6–8 20 40 Preimplantation Spontaneous Stillbirths Live births embryos abortions Incidence of aneuploidy 1–2% ~20% ~20% 35% 4% 0.3% Common Various 45, X, + 16 + 13, + 18, + 13, + 18, + 21 aneuploidies +21, +22 + 21 XXX, XXY, XYY The frequency decreases again by an order of magnitude, with about 0.3% of newborns being aneuploid.  Other Forms of Chromosome Changes Uniparental disomy A condition in which both copies of a chromosome are inherited from a single parent Copy number variation A situation in which a particular gene or chromosomal region is present in multiple copies Fragile sites Appear as gaps or breaks in chromosome-specific locations Gene Imprinting A small proportion (