Cytogenetics Course Nov 2022 - PDF

Chromosomal aberrations Cytogenetics Pr. Raouf Alami CM1 Module : Cellular and molecular biology Course: Molecular biology www.um6s...

Chromosomal aberrations Cytogenetics Pr. Raouf Alami CM1 Module : Cellular and molecular biology Course: Molecular biology www.um6ss.ma Année Universitaire: 2022 - 2023 1 1. Objective of the course If a chromosome pair loses or gains a member, or even part of a member, the delicate balance of the human body may be disrupted. In this course, we’ll examine how changes in chromosome number and structure come about, and how they can affect human health. At the end of the courses we will try to have an overview of some technics used for the detection of these abnormalities FACULTY OF MEDICINE – UM6SS -CASABLANCA  Introduction Approximately 2 meters of DNA if stretched end-to-end in each human cell Most of our cells contain 46 chromosomes Normal human cell contains 46 chromosomes, except for: 1. enucleate cells (i.e., red blood cells), 2. cell fragments (platelets), and 3. haploid germline cells (eggs and sperm), which contain only 23 chromosomes. These chromosomes carry thousands of genes, which tell your body how to develop and which keep it functioning from moment to moment during your lifetime 3  Introduction Chromosomal aberration A chromosomal abnormality, or chromosomal aberration, is a disorder characterized by a morphological or numerical alteration in single or multiple chromosomes, affecting autosomes, sex chromosomes, or both. Chromosomal abnormality Chromosomal aberration A disorder 1. morphological 2. numerical alteration in single or multiple chromosomes, 3. affecting autosomes, 4. sex chromosomes, or both.  Introduction The normal human karyotype contains DNA organized into 46 chromosomes: 22 pairs of homologous autosomal chromosomes and a set of sex chromosomes that compromise two X chromosomes in females or an X and a Y chromosome in males.  Chromosomes Telomere Centromere P P P q q q q Metacentric Acrocentric Sub-Metacentric Telocentric Types of chromosomes Each chromosome is composed of two sister chromatids Homologous chromosomes Sister chromatides Each chromatid represents one molecule of DNA 6  Groups of chromosomes Chromosomes are divided into seven groups 7 groups 7  Introduction Chromosome abnormalities usually involve an error in cell division (mitosis or meiosis), When chromosomal abnormalities occur in the prenatal, postnatal, or preimplantation periods, they have significant clinical consequences, i.e., : 1. spontaneous abortions, 2. stillbirths, ( death or loss of a baby before or during delivery) 3. Neonatal death/hospitalization, (when a baby dies in the first 28 days of life) 4. Malformations, (A structural defect in the body due to abnormal embryonic or fetal development) 5. Intellectual disability, (Involves problems with general mental abilities ; such as learning, problem solving, judgement) and daily life activities (communication and independent living) 6. Identifiable syndrome Accurate identification of these chromosomal errors is essential for prevention strategies, genetic counseling, and appropriate treatment.  Two basic categories of chromosomal abnormalities N >>>> S A. Numerical disorders are more common than structural ones A. A deviation from the normal diploid number B. missing chromosomes or duplicated chromosomes C. whole haploid sets of chromosomes may be added or lost. B. Structural abnormalities : the genomic rearrangement of one or more chromosomes. Usually : unequal exchange between chromosomes Exemples: deletions, translocations, and inversions, duplications, ring chromosomes, and isochromosomes.  Chromosomal abnormalities (continued) Chromosomal abnormalities may also classify as constitutional or acquired. 1. Constitutional chromosomal abnormalities arise during gametogenesis or early embryogenesis and affect all or a major portion of an organism’s cells. Their estimated incidence is around 20% to 50% of all human conceptions (more than a thousand different anomalies in liveborn patients) 2. Acquired chromosomal abnormalities typically develop during adulthood and affect a single clone of cells with a determined distribution in the body exclusively. These alterations are involved in the pathogenesis of many neoplasms, =linked to the development of neoplasms (tumors or cancers) and pathologies  A. Numerical chromosomal disorders 1. Euploidy 2. Aneuploidy (Heteroploidy) Occur in exact multiples of the Deviation from the diploid number of haploid number (n), chromosomes : 2n + 1, 2n -1 etc. Up to 3% of all human conceptions The gain and loss of one or two chromosomes, e.g. monosomy (2n-1), Example, triploidy (three sets of trisomy (2n+1), nullisomy (2n-2). chromosomes or 3N) Nullisomy =Loss of 2 homologous ch. all polyploidies 5 to 10% of all pregnancies monosomy, triploidy, is the leading genetic cause of miscarriage tetraploidy, and congenital defects. pentaploidy, hexaploidy, and so on) Most aneuploidies are lethal; nonetheless, there is a scarce amount of viable Are not life compatible with humans. syndromes.  Euploidy A. Numerical chromosomal disorders Euploidy is a chromosomal variation that involves the entire set of chromosomes in a cell or an organism. Euploidy is more tolerated in plants than in animals. There may be a : ✓single set (monoploidy), ✓two sets (diploidy), or ✓multiple sets (polyploidy, i.e. triploid, tetraploid, pentaploid, hexaploid, etc.) Exemple of euploidy : Triploidy A. Numerical chromosomal disorders The most common polyploidy is Triploidy carytype triploidy characterized by the 69,XXX presence of 3 groups of haploids. 69, XXX, 69, XYY 69, XXY Exemple of euploidy : Triploidy A. Numerical chromosomal disorders Human triploids 69 chromosomes rather than the normal 46 chromosomes per cell. Lethal or death early in the newborn period About 1 – 2% of all human conceptions may be triploid The live birth rate is 1:2500 Mosaicism occurs : may live for a while associated with profound retardation and physiological abnormalities 15  Mechanisms of Polyploidy A. Numerical chromosomal disorders How does it happen? Polyploids arise when a rare mitotic or meiotic catastrophe, such as nondisjunction, causes the formation of gametes that have a complete set of duplicate chromosomes. Diploid gametes are frequently formed in this way. When a diploid gamete fuses If a diploid gamete fuses with with a haploid gamete another diploid gamete a triploid zygote forms, a tetraploid zygote, 16 Mechanisms of Polyploidy A. Numerical chromosomal disorders Digyny vs Diandry Triploidy may be the result of either 1. Digyny (extra haploid set from mother) 2. Diandry (extra haploid set from father) = diplospermy / dispermy https://en.wikipedia.org/wiki/Digyny  Mechanisms of Polyploidy Digyny or Anomalies digyniadeisnombre the Diandry : fertilized by : 2 sperm fertilization of an diploid or by a diploid sperm, ovum by a monoploid making the fertilized egg sperm. triploid, resulting in early The result of digyny is a miscarriage triploid egg. (1 in 50,000) Diplospermy Dispermy ♀♀♂ ♀♂♂ 69,XMXPXP diplospermie II Digyny Diandry 69,XMYY diplospermie II 69,XMXPY diplospermie I Aneuploidy A. Numerical chromosomal disorders Having missing or extra chromosomes Nondisjunction : mistake in cell division where chromosomes do not separate properly in anaphase Usually in meiosis and occasionally in mitosis In meiosis it occurs during anaphase I or II Autosomal aneuploidy Sex chromosomes aneuploidy abnormal number of individuals have an abnormal autosomal chromosomes number of sex chromosomes.  Nondisjunction Normal 1st & 2nd meiotic division Two types of non-disjunction 1st meiotic 1st meiotic division division 2nd meiotic division 2nd meiotic division  Mitosis and Meiosis Mitosis results in two cells that are duplicates of the original cell. One cell with 46 chromosomes divides and becomes two cells with 46 chromosomes each. This kind of cell division occurs throughout the body, except in the reproductive organs. This is the way most of the cells that make up our body are made and replaced. Meiosis results in cells with half the number of chromosomes, 23, instead of the normal 46. This is the type of cell division that occurs in the reproductive organs, resulting in the eggs and sperm. 21  Meiosis (Reminder) Meiosis I = homologous chromosomes are seprated and then reducig the chromosome number by half Meiosis II = sister chromatids are separated producing 4 haploid gametes 22  Mitosis Non-disjunction in body cells When Non-disjunction happens during mitosis, chromosome number changes in body cells will not be passed on to children Mitotic nondisjunction can cause cancer 23  Aneuploidy A. Numerical chromosomal disorders How does it happen? There are two types of non-disjunction Non-disjunction of chromosomes Disorders of chromosome number are caused by nondisjunction, which occurs when pairs of homologous chromosomes or sister chromatids fail to separate during meiosis I or II (or during mitosis). 24 Exemple of Aneuploidy : Monosomy/Trisomy A. Numerical chromosomal disorders Two common types of aneuploidy have their own special names: Monosomy is when an organism has only one copy of a chromosome that should be present in two copies (2n−1). Trisomy is when an organism has a third copy of a chromosome that should be present in two copies (2n+1) 25 Exemple of Aneuploidy A. Numerical chromosomal disorders Larger numbers Aneuploidy also includes cases where a cell has larger numbers of extra or missing chromosomes, as in (2n−2),(2n+3),etc. 26 Genetic disorders caused by aneuploidy A. Numerical chromosomal disorders 1. Autosome chromosomal aneuploidy Most human autosomal monosomies are lethal Because on an imbalance in the chromosome product Smaller chromosomes ((13, 15, 18, 21, or 22) can allow individual to survive (with some disabilities) Imbalance of the gene products 27 Exemples of Trisomy of Autosomes A. Numerical chromosomal disorders 1. Autosomal chromosomal aneuploidy Chromosomes are divided into seven groups 21 13 18 =GDE=DPE 1. Trisomy 21 or G-trisomy (Down syndrome) 2. Trisomy 13 or D-trisomy (Patau syndrome) 3. Trisomy 18 or E-trisomy (Edward syndrome) Exemple 1 of aneuploidy A. Numerical chromosomal disorders 1. Autosome chromosomal aneuploidy Trisomy 21 The most common trisomy among embryos that survive to birth is Down syndrome, or trisomy 21. People with this inherited disorder have sort of characteristics as facial distinctions, developmental delays, etc. 47,XX,+21 (female) or 47,XY,+21 (male) About 1 in every 800 newborns is born with Down syndrome. the likelihood that a pregnancy will result in an embryo with Down syndrome goes up with a woman's age, particularly above 40years. This is probably because of more frequent nondisjunction in the developing eggs of older women. 29 Down syndrome A. Numerical chromosomal disorders 1. Autosome chromosomal aneuploidy Trisomy 21  Exemple 2 of aneuploidy (Patau Syndrome) A. Numerical chromosomal disorders Trisomy 13 1. Autosomal chromosomal aneuploidy 1st described by Bartholin (1657) & redefined by Patau (1960). Chromosomal complement: 47,XX,+13 (female) or 47,XY,+13 (male) Phenotype: Male or female Incidence: 1:12,000 (increases with the age of mother) Half of babies born with Trisomy 13 live longer than two weeks Fewer than 10% will survive the first year of life. Approximately 13% survive until 10 years of age Exemple 2 of aneuploidy (Patau Syndrome) A. Numerical chromosomal disorders Trisomy 13 1. Autosome chromosomal aneuploidy Congenital heart defects Renal tract anomalies Mental deficiency Low birth weight Abnormal development of frontal lobe Absence of corpus callosum Hypoplasia of cerebellum Sloping forehead Scalp defects Exemple 3 of aneuploidy (Edward Syndrome) A. Numerical chromosomal disorders Trisomy 18 1. Autosome chromosomal aneuploidy Chromosomal complement: 47,XX,+18 (female) or 47,XY,+18 (male) Phenotype: Male or female Incidence: 1: 8000 Between 60% and 75% survive to their first week. Between 20% and 40% survive to their first month. No more than 10% survive past their first year Exemple 3 of aneuploidy (Edward Syndrome) A. Numerical chromosomal disorders Trisomy 18 1. Autosome chromosomal aneuploidy Mental deficiency Growth retardation Short sternum Micrognathia LOWER JAW UNDERSIZE Renal anomalies  2. Sex chromosome aneuploidies SCA >>> A A A. Numerical chromosomal disorders Sex chromosome neuploidies are better- tolerated than autosomal ones. Why ? because human cells have the ability to shut down extra X chromosomes in a process called X- inactivation, or XCI (X-chromosome inactivation) The purpose of the X-inactivation system is to shut down the second X of an XX female It can also shut down more X chromosomes if they are present. A Barr body is an inactive X chromosome Most of the genes on the Barr body are not transcribed. X-inactivation is a random process. It happens during embryonic development. 35 Sex chromosome aneuploidies A. Numerical chromosomal disorders 3 examples of X chromosome aneuploidies Klinefelter syndrome Triple X syndrome Double Y syndrome 36 Sex chromosome aneuploidies 1. Klinefelter syndrome, A. Numerical chromosomal disorders Phenotype: Male XXY Males have an extra X chromosome (XXY genotype). Klinefelter syndrome may involve several extra Xs, leading to an XXXY or XXXXY genotype.) Klinefelter syndrome is thought to affect 1 out 47,XXY of every 500 male newborns In general, life expectancy is normal. 37 Sex chromosome aneuploidies 1. Klinefelter syndrome, A. Numerical chromosomal disorders Features of Klinefelter Syndrome Affected men may be infertile or develop less dense body Tall stature; thin build; long lower limbs Testicular atrophy Infertility (aspermatogenesis) Low level of intelligence Sex chromosome aneuploidies 2. Triple X Syndrome (Superfemale) A. Numerical chromosomal disorders Called trisomy X Phenotype: Female XXX Woman has an X: XXX genotype about 1 out of every 1,000 female newborns. Most females with triple X syndrome have normal sexual development and are able to conceive children. 47,XXX Girls and women with triple X syndrome can lead normal lives. Sex chromosome aneuploidies 2. Triple X Syndrome (Superfemale) A. Numerical chromosomal disorders Women with an XXX genotype have female sex characteristics are fertile In some cases, triple X syndrome may be associated with learning difficulties, late development of motor skills in infants, and problems with muscle tone Mother-of-five, Prahnee Smith Sex chromosome aneuploidies A. Numerical chromosomal disorders 3. Double Y Syndrome Phenotype: Male XYY XYY syndrome is a genetic condition in which a male has an extra Y chromosome. Normal in appearance : There are usually few symptoms. taller than average, an increased risk of learning problems. 47,XYY The person can be fertile Aggressive behaviour he median age of survival was 77.9 years for controls and 67.5 years for 47,XYY persons, Frequency: ~1 in 1,000 males Sex chromosome aneuploidies Monosomies of Chromosomes A. Numerical chromosomal disorders Presence of only one member of a chromosome pair in a karyotype More damaging than equivalent trisomy Can involve autosomes or sex chromosomes : Autosomal monosomies usually abort spontaneously Monosomy of X chromosome results in XO condition called Turner syndrome Sex chromosome aneuploidies Turner Syndrome Monosomies of Chromosomes: A. Numerical chromosomal disorders Turner syndrome Phenotype: Female Incidence: 1:5000-8000 45,XO  Monosomies of Chromosomes: A. Numerical chromosomal disorders Turner syndrome Short statured female Sterility Short, webbed neck Prominent ears with defective hearing A small chin and jaw Defective vision  B- Structural Chromosomal Abnormality (CSA) => breakage + reconstitution Structural Chromosomal Abnormality Results from chromosome breakage Followed by reconstitution in an abnormal combination Breaks in any chromosome may be induced by various factors Structural abnormalities are when part of an individual chromosome is missing, extra, switched to another chromosome, or turned upside down. Chromosomal abnormalities can occur as an accident : 1. during the formation of the egg or the sperm, or 2. during the early developmental stages of the fetus. gametogenisis + early foetogenisis  B- Structural Chromosomal Abnormality (CSA) Structural rearrangements are defined As : Balanced 1. if the complete chromosome set is still present. No gain No loss Unbalanced 2. if there is additional or missing information. Unbalanced rearrangements include deletions, duplications. Serious negative effects 46  B- Structural Chromosomal Abnormality (CSA) 1. Deletion Lost of genetic material Ionizing radiation Deletion: occur when a chromosome breaks and some genetic material is lost. Deletions can be large or small, and can occur anywhere along a chromosome: 1. A terminal deletion is the loss of the end of a chromosome. Terminal deletion 2. An interstitial deletion results after two breaks are Interstitial deletion induced if the terminal part (AB) rejoins the main body of the chromosome, with the acentric At the centromere fragment (CD) being lost. Involves 1 Chromosome  B- Structural Chromosomal Abnormality (CSA) 1. Deletion Isochromosome 3. Deletion at the centromere : when the centromere is divided perpendicularly to the long axis of the chromosome. An isochromosome is created 48  B- Structural Chromosomal Abnormality (CSA) 1. Deletion Ring chromosomes Ring chromosomes : usually occur when a chromosome breaks in two places, typically at the ends of the p and q arms, and then the arms fuse together to form a circular structure. The ring may or may not include the centromere, depending on where on the chromosome the breaks occur. In many cases, genetic material near the ends of the chromosome is lost. Caryotype with a ring at the chromosome 13 in female. 46,XX, r(13) Involves 1 Chromosome Deletions in Humans Examples of chromosomal deletion syndromes include Cri-du-chat syndrome Micro deletion of chromosome 5 Di-George syndrome Micro deletion of chromosome 22 Schizophrenia & Obsessive Compulsive Disorder Micro deletion of chromosome 22 associated Angelman syndrome Micro deletion of chromosome 15 Prader-Willi syndrome Micro deletion of chromosome 15 Cri-du-chat syndrome or 5p minus Syndrome Caused by missing piece from the p arm on the chromosome 5 1st autosomal deletion described Characteristic cat-like cry, which disappears with age poor muscle tone Severe mental retardation Behavioral problems Excessive drooling = saliva 46,xx,del5p14.2. For more detail on this abnormality : https://fivepminus.org/about-5p-syndrome/  B- Structural Chromosomal Abnormality (CSA) 2. Inversion Inversion occurs when a chromosome breaks in two places; the resulting piece of DNA is reversed and re-inserted into the chromosome. (180°) Depending on the breakage sites the inversion would be : 1. Paracentric : An inversion that does not involve the centromere (Occurs in the long (q) arm or short (p) arm) 2. Pericentric = An inversion that includes the centromere Involves 1 Chromosome B- Structural Chromosomal Abnormality (CSA) 3. Duplication Duplications : Occur when part of a chromosome is abnormally copied (duplicated). This type of chromosomal change results in extra copies of genetic material Involves 1 Chromosome B- Structural Chromosomal Abnormality (CSA) 4. Insertion or SUBSTITUTION A segment of one chromosome is translocated and inserted into an interstitial region of another non-homologous chromosome Involves 2 Chromosomes  B- Structural Chromosomal Abnormality (CSA) 5. Translocation Translocation is a type of chromosomal abnormality in which a chromosome breaks and a portion of it reattaches to a different chromosome. Robertsonian translocation Reciprocal translocation 55  B- Structural Chromosomal Abnormality (CSA) 5. Translocation 5.1. Robertsonian translocation p arm Centromere A Robertsonian translocation is a type of translocation involving two homologous (paired) or non-homologous chromosomes Robertsonian translocations occur in the five acrocentric chromosome pairs (chromosome pairs where the short arms are fairly short), namely 13, 14, 15, 21 and 22. q arm The participating chromosomes break at their centromeres and the long arms fuse to form a single, large chromosome with a single centromere. -small chromosomes => 13,15,18,21,22 -acrocentric => 13,14,15,21,22 2n= 45t(13,21) Long arms of 13 and 21 fused to Overview of form a single chromosome. chromosome 13 Usualy non consequences cause the lost regions are repetetive ribosomal genes 13 21 Involves 2 Chromosomes For more details on the chromosomal transmision http://atlasgeneticsoncology.org/Educ/PolyMecaEng.html  B- Structural Chromosomal Abnormality (CSA) 5.Translocation 5.2. Reciprocal translocation Reciprocal translocation is a form of gene rearrangement where portions of two chromosomes are simply exchanged with no net loss of genetic information. 1. Involves 2 chromosomes 2. One segment breaks on each chromosome 3. The 2 chromosomes exchange broken segments Possible consequences: 1. Alteration of the structure of the genes( in new location) Involves 2 Chromosomes and /or 1. Abnormal expression of the translocated gene(s). Summary of Chromosomal structural abnormalities (CSA) CSA involving 2 chromosomes CSA involving one chromosome https://www.genome.gov/about-genomics/fact-sheets/Chromosome-Abnormalities-Fact-Sheet Chromosomal anomalies in spontaeous abortions abnormalities Reported rate occurrence (%) Autosomal trisomy 50-60 Polyploidy 20-25 Monosomy X 10-20 translocations 2-5 https://basicmedicalkey.com/chromosomal-abnormalities/ 59 Quiz 60  How How do we study the chromosmes’ abnormalities? Cytogenetics 61  Cytogenetics Introduction Cytogenetics involves the study of chromosomes Both the number and structure of the chromosomes are analyzed Cytogenetics is the branch of genetics that studies the structure of DNA within the cell nucleus. This DNA is condensed during cell division and form chromosomes. The cytogenetic studies the number and morphology of chromosomes. Using chromosome banding techniques (classical cytogenetics) or hybridization fluorescently labeled probes (molecular cytogenetics). Cytogenetics What tissues are appropriate for chromosome study? Any tissue that can be stimulated to undergo cell division in vitro When can we see the chromosomes ? : During miTosis With what? Light microscope Some examples of studied cells: Chorionic villi (placental tissue) Amniotic fluid Perifiral blood (lymphocytes) Skin (fibrobalsts Bone marrow 63  Comparison of technical details between conventional cytogenetic and major molecular approaches Abbreviations: FISH, fluorescence in situ hybridization; CGH, Comparative genomic hybridization. Techniques Method Characteristics Application Advantages Conventional Genome wide Special dye generate Detection of numerical cytogenetics screening for Cell culturing banding pattern for each and structural (G-banding, R- chromosome level chromosome chromosomal anomalies banding) abnormalities Use probe to search for Detect all types of Interphase Conventional Molecular target sequences in balance and unbalanced cytogenetics possible FISH technique chromosome defects Simple procedures Spectral Arresting cell in Allows the painting of Detection of complex Fast characterization karyotyping metaphase every chromosome anomalies of euchromatic Comparative Whole genome wide Identify and assess hybridization of screening of genomic Molecular biomarkers CGH differentially labeled anomalies technique total genomic tumor Gene discovery, No need for cell DNA and reference DNA functional analysis culture Identification Molecular use of specific DNA aneuploidy, deletions, Array-CGH High-resolution technique binding proteins duplications or amplifications Cytogenetics Conventional technics 1. Cells are stimulated to divide 2. Incubated for 48 to 96 hours 3. Cells are arrested at mitosis (Colchisin that prevents the elongation of the microtubule polymer) 4. Cells are dropped onto glass slides and G banded 65  Cytogenetics How is it possible to differenciate between chromosomes? Chromosome banding chromosome banding refers to alternating light and dark regions along the length of a chromosome, produced after staining with a dye. A band is defined as the part of a chromosome that is clearly distinguishable from its adjacent segments by appearing darker or lighter with the use of one or more banding techniques. Banding types Dye DNA sequence Where Q-banding quinacrine AT – rich DNA Chro. arms G-banding Giemsa AT – rich DNA Chro. arms Different R-banding CG-rich DNA Chro. arms techniques Different Centromere C-banding AT-rich DNA 66 techniques Distal Y chro.  Cytogenetics Chromosome banding Chromosome banding refers to alternating light and dark regions along the length of a chromosome, produced after staining with a dye. A standard metaphase karyotype (450–550 bands) is useful for identifying extra or missing chromosomes, High-resolution banding (550–800 bands) is effective at identifying more subtle structural abnormalities including deletions, duplications, translocations, and inversions. A diagram recording the distribution of dark bands in human chromosomes 67 Cytogenetics Karyotyping technique Overall scheme for the production of metaphase chromosomes for traditional cytogenetic analysis. 68  Cytogenetics Giemsa staining protocole and principle Metaphase Trypsin (partially digest chromosomes the chromosome) Stained with Giemsa (methylene blue and eosin) 92% of the human genome is euchromatic Heterochromatin Euchromatin (AT-rich) DNA (GC-rich) Condensed Less condensed Relatively gene-poor, Transcriptionally active Incorporates Giemsa Incorporates less Giemsa Light bands Dark bands 69  Cytogenetics Giemsa staining Banding Patterns G-banding allows each chromosome to be identified by its characteristic banding pattern. The banding pattern can distinguish chromosomal abnormalities or structural rearrangements, such as: 1. translocations, 2. deletions, 3. insertions, and 4. inversions. The short arm is p, The long arm is q. Each arm is divided into larger regions that are An example of G-banding pattern further subdivided into smaller bands and on X-chromosome interbands.  Cytogenetics Reporting cytogenetics results Cytogenetic analysis provides a genome-wide snapshot of an individual's chromosomes by the process of pairing and arranging all of them in an order, Can also reveals changes in chromosome numbers (aneuploids) and structural changes The karyotype is the representation obtained by microphotography of the morphological appearance of all the chromosomes of a metaphase cell. Karyotyping is the process of pairing and ordering all the chromosomes of an organism. Two possible presentations The karyotype has become a routine medical prescription for certain pathologies. Optical microscopy 71  Cytogenetics Reporting cytogenetics results What are the Similarities Between Karyotype and Idiogram? The karyotyping technique gives both karyotype and ideogram. Both show the total number of a cell. Moreover, they show the morphological features of the chromosomes. In both pictures, chromosomes are ordered in a series of decreasing size. 72  Cytogenetics Karyotype nomenclature The International System for Human Cytogenetic Nomenclature (ISCN) is an international standard for human chromosome nomenclature, include band names, symbols and abbreviated terms used in the description of human chromosome and chromosome abnormalities. How to Write a Notation of a Karyotype Numerical chromosomal disorders 1. Count the number of pairs of chromosomes in the karyotype, except the sex chromosomes, the last two in the set. Write this number. In a normal human, the number will be 46. 2. Determine the sex chromosomes, whether they are "XX" or "XY." 3. Write this combination next to the number after a comma. In a normal woman, this will look like this "46, XX) 4. If the karyotype has an extra 21st chromosome, write (47, XX, +21) If there is an extra sex chromosome, write 47, then the sex chromosomes; for example, "47, XXX." Cytogenetics Karyotype nomenclature Structural chromosome aberrations Structural chromosome aberrations are the result of chromosome breakage and abnormal reunion of broken chromosomes. chromosome breakage abnormal reunion of broken chromosomes. karyotype for Cri du Chat Syndrome would be: 46,xx,del5p14.2. ✓ There is 46 chromosomes (23 pair); ✓ “xx” in this case means it is a girl with two “x” chromosomes ✓ del5p indicates that there is a deletion on the 5th chromosome, ✓ “p” arm. ✓ the 14.2 is the location on the “p” arm that the deletion has occurred. 74 Cytogenetics Important dates 1956: discovery of hypotonic shock Male: 46 chromosomes (Tjio and Levan) 1959-69: number anomalies: Trisomy 21 (Lejeune 1959) 1970: discovery of band marking 1981: FISH technique using a labeled probe (radio activity) 1986: discovery of fluorescences! (no radio activity)  Modern method of Karyotyping Fluorescence in- situ hybridization (FISH) =probe is a small piece of DNA or RNA used to find a specific (FISH) is used for karyotyping sequence in a sample. The fluorescent probes are nucleic acid labeled with fluorescent groups and can bind to specific DNA/RNA sequences. It was developed in the early 1980s. Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes Preparing FISH Probes 76  Modern method of Karyotyping Fluorescence in- situ hybridization (FISH) Probe preparation Patient’s cell FISH is based on the use of chromosome region-specific and fluorescent-labeled DNA probes. pieces of genomic DNA that can Probes detect their complementary DNA sequences The probe is labeled : produce a fluorescent signal detection of small genomic alterations of 50 Kb to 100 Kb direct visualization of these alterations Basic steps of fluorescent in situ hybridization technique 77 Modern method of Karyotyping Fluorescence in- situ hybridization (FISH) Karyogram from a human female lymphocyte probed for the Alu sequence using FISH https://en.wikipedia.org/wiki/Karyotype#/media/File:PLoSBiol3.5.Fig7ChromosomesAluFish.jpg 78  2. Molecular cytogenetics To overcome the limitations of banding analysis: Molecular cytogenetic techniques such FISH, spectral karyotyping (SKY), comparative genomic hybridization (CGH) have emerged as successful diagnostic tools. 79  Comparison of technical details between conventional cytogenetic and major molecular approaches Techniques Method Application Advantages Conventional Detection of numerical and cytogenetics (G- Cell culturing structural chromosomal Genome wide screening banding, R- anomalies banding) Detect all types of balance Conventional FISH Molecular technique Simple procedures and unbalanced defects Spectral Arresting cell in Detection of complex Different chromosomes karyotyping metaphase anomalies different color Identify and assess Whole genome wide biomarkers screening CGH Molecular technique Gene discovery, functional No need for cell culture analysis Identification aneuploidy, Array-CGH Molecular technique deletions, duplications or High-resolution amplifications Abbreviations: FISH, fluorescence in situ hybridization; CGH, Comparative genomic hybridization. Resolution to detect gaisn or losses Hight generation sequencing Singer sequencing 1bp SNP 50 - 100K bp a-CGH 50 - 100K bp FISH 100 - 500K bp G- banding 3 à 5 Mbp 81 82  Spectral karyotyping The innovation of SKY is that it gives each chromosome a different color, so that it's easy to determine which is Chromosome 1 and which is Chromosome 18, so it's much easier for researchers and clinicians to figure out abnormalities of the chromosomes. 83 Microarray-based Comparative Genomic Hybridization (aCGH) Schena et al., 1995 Microarray analysis, is a new cytogenetic technology Iidentify small deletions and duplications higher resolution than traditional karyotyping. Combines the principles of CGH with the use of microarrays. 84 References https://arup.utah.edu/media/andersen-introCyto2-2020/lecture-slides.pdf http://www.kjcls.org/journal/view.html?doi=1 0.15324/kjcls.2018.50.4.375 https://www.genome.gov/genetics- glossary/Spectral-Karyotype 85