Human Genome Lab 1: Principles of Cytogenetics (Karyotyping) PDF

Course: Human Genome Lab 1: Principles of Cytogenetics (Karyotyping) Karyotyping Definition The karyotype is the characterization of number, size, and morphology of the set of chromosomes of a species, as seen under the microscope. Then arrange it into homologues pairs, in descending order ac...

Course: Human Genome Lab 1: Principles of Cytogenetics (Karyotyping) Karyotyping Definition The karyotype is the characterization of number, size, and morphology of the set of chromosomes of a species, as seen under the microscope. Then arrange it into homologues pairs, in descending order according to their size. In Conclusion: The karyotype is the arrangement of metaphase chromosomes in homologous pairs according to size and location of centromere. Metaphase chromosome anatomy Short Arm (p) Why do we extract metaphase chromosomes in karyotyping ? This stage of the cell cycle in which the Centromere chromosomes assume their characteristic condensed, discrete shape. Long Arm (q ) Two sister chromatids joined by the centromere  Chromosome Morphology Chromosomes has 4 main morphologies:  Important Definition Homologues Chromosomes: This means the pair of chromosomes that are structurally identical. Each homolog come from each parent ie. One homolog comes from the mother and the other comes from the father. Sister Chromatid : either of the two identical chromatids that are formed by replication of a chromosome during the S phase of the cell cycle, are joined by a centromere and segregate into separate daughter cells. Applications of Karyotyping It is used to detect chromosomal aberrations like deletion, duplication, translocation, and non-disjunction of chromosomes. It helps to identify the abnormalities of chromosomes like aneuploidy. Karyotyping of the foetus helps to detect the birth defects (e.g. Amniocentesis). It is also used in predicting the evolutionary relationships between species. ( Comparative Karyotyping )  How to obtain metaphase chromosomes Somatic cell culturing Embryonic cell sample Cells from amniotic fluid from any tissue e.g. after IVF i.e. amniocentesis tumors  How to obtain metaphase chromosomes Embryonic cell sample after IVF (Preimplantation genetic diagnosis PGD) PGD uses IVF, in which multiple eggs are matured and retrieved. The oocytes — or primitive egg cells — are inseminated with a single sperm using intracytoplasmic sperm injection. The resulting embryos are grown in culture until the six-to-eight-cell stage, which is day three of embryo development. At this point, the embryo is biopsied with the removal of one to two cells. This process does not damage the cells remaining within the embryo.  How to obtain metaphase chromosomes Cells from amniotic fluid i.e., amniocentesis Amniocentesis is a prenatal test that can diagnose genetic disorders (such as Down syndrome and spina bifida) and other health issues in an unborn baby. Amniotic fluid is removed from the uterus for testing or treatment. Amniotic fluid is the fluid that surrounds and protects a baby during pregnancy. This fluid contains foetal cells and various proteins. Experiment (1) Extraction of Metaphase chromosomes from femur of albino mouse  Extraction of Metaphase chromosomes from femur of albino mouse Procedures​ Colchicine treatment​ 1.Inject the animals (mice or rat) with colchicine (4mg/kg) 1.5-2 hrs before killing by cervical dislocation.​ Note that: Kill mouse by cervical dislocation. Why? To avoid bleeding that affect bone marrow cells. Extraction of bone marrow 1. Cut out femur bone and clean it from the surrounding tissues. 2. On a filter paper, cut away the epicondyle tips. 3. Gently inject 3-4 ml of physiological salt solution (0.9% sodium chloride) into one end of the femur bone to extract bone marrow into centrifuge tube at the other end of the bone. 4. Use a clean pasteur pipette to suspend the bone marrow cells in the saline solution. 5. Centrifuge for 5 mins at 1000rpm the discard the supernatant and disrupt cell pellet.  Hypotonic treatment 6. Suspend cell pellet in 3ml of 0.75M KCl sol. (5.592gm KCl/1000ml H2O) and mix gently. 7. Incubate the tube at 37˚C for 10-15 mins. 8. Centrifuge again for 5 mins at 1000 rpm, then discard the supernatant and disrupt cell pellet.  Fixation 9. Add 2-3ml of freshly prepared methanol-glacial acetic acid (3:1) to the cell pellet, 1 drop at a time, mix gently and let stand for 10mins at room temperature. 10. Centrifuge for 5mins at 1000rpm, discard the supernatant and disrupt the cell pellet. 11. Fix again by adding 3ml of the fixative, mix, let stand for 10mins at room temperature. 12. Centrifuge, discard supernatant and disrupt cell pellet. 13. Fix for the third time by repeating steps 11&12. 14. Finally, add 1-2ml of freshly prepared fixative to the cell pellet to give a condensed cell suspension.  Spreading and flame drying 15. To prepare chromosomes for microscopic examination, drop 3-5 drops of the cell suspension on a clean wet glass slide previously kept at 4˚C in 70% ethanol. 16. Close the glass slide to the flame of benzene burner to burn the alcohol and causes the bursting of the cells on the slide.  Staining Procedure 1. Prepare three clean staining jars, the first containing buffered Giemsa solution, while the second and the third jars containing buffer solution in order to remove excess stain. 2. Arrange the dry labelled slides in the first jar, stain for 30-5 minutes 3. Rinse the stained slides in the second and third jars for 10 minutes each 4. Allow slides to dry on filter paper 5. Coverslip with glass cover using a suitable mountant Mention the role of Cholchicine, cervical dislocation, 0.9 NaCl, Kcl, Methanol-glacial acetic acid, Flame- burner, and Giemsa in the previous procedure. Principles of Karyotyping 1. Chromosomes are serially arranged by common sense in homologous pairs according to their decreasing size. 2. If 2 Chromosomes are apparently equal in size, arrange according to shape. 3. Chromosomes are positioned in 4 rows: A Row for the large-sized Chromosomes. (gp. A&B ) A Row for the medium-sized Chromosomes. (gp. C) A Row for the small-sized Chromosomes. (gp. D&E) A Row for the minute-sized Chromosomes. (gp. F&G)  Note: Human karyotype - set of 46 chromosomes (number – species specific characteristic) 22 homologous pairs of autosomes and 1 pair of sex chromosomes arranged and numbered according to the chromosome size and type (centromere position) and banding pattern into homologous pairs and seven chromosome groups Group A (1-3) – large metacentric chromosomes (but chr. 2 – submetacentric) Group B (4-5) – large submetacentric Group C (6-12, X) – medium-sized submetacentric Group D (13-15) – medium-sized acrocentric Group E (16-18) – short submetacentric Group F (19-20) – short metacentric Group G (21-22, Y) – short acrocentric (but chr. Y – submetacentric)  Arranged in descending order acc. to size Arranged into 4 rows Sorted into 7 groups according to their size and shape 4. For uniformity purposes, all centromeres of the chromosome pairs of a row must be adjusted to the same horizontal level. 5. Every chromosome in positioned so that the short arms are directed up wards & long ones are directed downwards. 6. Sex chromosome are positioned according to one of the following rules: Both X & Y are put together at the end of the last row of the karyotype 7. Each chromosome pair should take a serial number which is usually written rig below the pair wherever it is positioned. Centromeres Short are placed arm directed on the line upward Each is given a serial The Sex no. chromosomes are arranged at the end Karyotype measurements  Karyotype measurements 1- Average length of short arms of chromosome no 1 of a pair. 2- Average length of long arms of chromosome no 1 of a pair. 3- Average length of short arms of chromosome no 2 of a pair. 4- Average length of long arms of chromosome no 2 of a pair. 5- Total length of chromosome no 1 a pair. 6- Total length of chromosome no 2 a pair. 7- Average Total length of chromosomes no 1& no 2 of a pair. 8- Centromeric index. 9- Arm ratio. 10- Relative length. Parameters: The parameters used in karyotype preparation are: (i) The number of chromosomes, (ii) The length of each chromosome, (iii) The length of chromosome arms (both short and long arms), (iv) The position of centromere, (v) The existence and localization of secondary constriction, (vi) The position and size of the satellites, (vii) Absolute and relative chromosomal size, (viii) Basic chromosome number, (ix) The degree and distribution of heteromatic regions, (x) Amount and location of repeated sequence. Average length of short / long arms of Ch. no 1 of a pair 1 First: draw 2 diagonal lines marking the start and the Draw end of the arm Second: measure the distance between the 2 lines Measure and write it above the measured one Repeat Third: repeat the same steps for the second arm Fourth: calculate the average of the lengths by Calculate dividing their sum by 2 (Average Short arm Ch. No1 = ( Σ of length / 2)  Tabulate the calculations as following Shape​ R. Length​ A. R.​ C. I​ AV. Total Total AV. Short​ AV. Long​ Ch. No.​ ​ Length​ Length​ ​ 4 2=2/)2+2( 2=2/)2+2( 1st Ch.​ 4.5=2/)5+4( =2/)2+2.5( =2/)3+2.5( 5 2nd Ch.​ 2.25 2.75 Average length of short / long arms of Ch. no 2 of a pair 1 Measure Measure of pair 1 the short arms and long arm of Ch.2 Average Average the lengths Calculate Calculate the total length of chromsome Sum both total lengths of Ch. 1&2 then Sum average them Important calculations  Calculations Shape​ R. Length​ A. R.​ C. I​ AV. Total Total AV. Short​ AV. Long​ Ch. No.​ ​ Length​ Length​ ​ =2/4.5 1=2/2 4 2=2/)2+2( 2=2/)2+2( 1st Ch.​ 0.4 4.5=2/)5+4( 2.75/2.2 2.75/4. =2/)2+2.5( =2/)3+2.5( 5 2nd Ch.​ 1.2=5 0.6=5 2.25 2.75 Note that: The length of haploid set is measured either by: A) The summation of (Average total length) B) The summation of total length then divided by 2 Identification of the shape according to the CI or Arm Ratio  Irregularities Acrocentric and Telocentric chromosomes have minute short As shown here; the long arm has curvature, thus, it arms thus, they cannot be measured in cannot be measured by the usual way. the usual way. Solution: it's measured in 2 steps and then Solution: it's given a length of 0.001 summed together cm  Types of Karyotypes An Asymmetric karyotype is a karyotype which shows a larger difference between the smallest and largest chromosomes of the set. It has ewer metacentric chromosomes. Most of the chromosomes are acrocentric. Asymmetric karyotype is considered to be a relatively advanced feature. It has evolved through structural chromosome changes. In flowering plants, scientists have observed a predominant trend towards the asymmetric karyotype. Moreover, an increased asymmetric karyotype is associated with specialized zygomorphic flowers. Ginkgo biloba also has asymmetric karyotype A symmetric karyotype is a karyotype which shows a smaller difference between the smallest and largest chromosomes in the set. It consists of more metacentric chromosomes. All the chromosomes are approximately the same size. Moreover, they have media or sub-median centromeres. The symmetric karyotype is not considered an advanced feature compared to the asymmetric karyotype. In fact, it represents a primitive state.​ ​  Analysis of Chromosomal Aberrations in Bone marrow Cells  Chromosomal Aberrations Any change in the normal structure or number of the chromosomes, often results in physical or mental abnormalities. Structural abnormalities such as: Deletion Duplication Inversion Translocation  Deletion A mutation causing part of the chromosome to be missing.  Duplication A mutation causing part of the chromosome to be repeated, resulting in extra genetic material.  Inversion A mutation resulting in a portion of a chromosome being in the opposite orientation.  What Causes Chromosomal Aberration? Chemical Agents (EMS) Physical agents (X ray)  Clastogen Also known as Clastogenic agent. It is an agent that can cause structural changes in the chromosomes.  It can cause breaks in chromosomes that result in the gain, loss, or rearrangements of chromosomal segments. It can also cause sister chromatid exchanges (SCE), that occur during DNA replication.  Bone Marrow It is the soft, spongy, gelatinous tissue found in the hollow spaces in the interior of bones. Progenitor cell lines in the bone marrow produce new blood cells and stromal cells. It is also an important part of the lymphatic system.  Testing the Effect of Clastogen on Animal Model Such as: Chinese Mouse Rat Hamster  The Experiment Parameters Animal Treatment Control Test Control Treatment group group Sample sample Route of Dose & Sex weight Age Solvent administration Duration We select the suitable according to … Sex Same/Different response to the chemical according to the sex. Age Higher mitotic index in younger animals (6-8 weeks in mice & 10-12 weeks in rats). Weight Narrow range of weights to adjust the dose  Route of Administration Selected according to the target of the experiment. o Intra-peritoneal (I.P) is used to maximize the chemical exposure to the bone marrow. o Feeding or Drinking to resemble the effect on human. Solvent Selected according to the chemical nature: o Water soluble dissolved in Isotonic Saline. o Non- Soluble in water dissolved in organic solvent such as DMSO.  Dose and Duration The maximum dose should be selected according to: o Toxicity o Mitotic index o Chemical or physical limitations such as the drug solubility. o Cell cycle kinetics The appropriate interval between doses and time of exposure to the dose was determined according to the pre-determined Maximum dose: o One-third the maximum dose. o One-tenth the maximum dose.  The control group and control treatment The animals considered as the control should be treated by the same volume and route of administration & divided into: o Positive control is a control group in an experiment that uses a treatment that is known to produce results Negative control is a control group in an experiment that uses a treatment that isn't expected to produce results  Sampling times T0 (The time of starting the experiment) Samples obtained three times: 6 hours, 12 hours and 24 hours after T0 The animal pre-injected by colchicine to accumulate metaphase cells.  Obtaining Cells At the end of the experiment the animals are sacrificed at the appropriate interval by cervical dislocation. Both femurs are quickly removed, muscle is cleaned away from the bone The marrow forced out using a syringe mounted with 5 ml KCl solution into centrifuge tube and incubated at 37C for 20 minutes..  Hypotonic treatment Supernatant is removed by gentle aspiration unit a small volume remains above the pellet. Add potassium chloride (KCL) pre-warmed to 37oC drop-wise with agitation to approximately 5 ml. Incubate 20 min in 37 °C water bath.  Fixation Add 0.5 ml fixative buffer (3:1 absolute methanol: glacial acetic acid) made freshly immediately before using drop-wise with agitation. Allow to stand at room temperature for 15-20 min. Centrifuge near 800 rpm for about 4 min. Gently aspirate supernatant leaving a small volume over pellet. Resuspend cells in remaining volume. Repeat the steps several times.  Slide preparation Drop 1 or 2 drops of suspension onto a clean wet slide. Quickly blot sides and back of slide. Blow once across slide and place onto slide warmer to dry. Prepare a minimum of 2 slides per animal. Slides are immediately coded with number which has been correlated with the animal number. Stain with Giemsa stain, dry thoroughly, and apply coverslips.  Cell Selection By using Low magnification power the field of cells was selected, some cells were accepted for scoring and others rejected for some reasons: o Complexity of aberrations. o Cells not well spread, and the overlap of chromosomes prohibits an accurate analysis. o Non-chromosomal material such as: dirt or stain crystals. o Separation of chromatids (Anaphase) prevents an accurate analysis.  Scoring and classification of aberrations Aberrations classified into general categories: Chromatid-type deletions Chromosome-type deletions Chromosome translocation These aberrations recorded to Aberration score sheet.  Some typical chromosome aberrations on bone marrow cells of rats. chromatidic break ,chromatidic gap isochromatidic gap, deletion, fragments ,micronucleus.  Scoring and classification of aberrations The results for any sample should be presented as: o The aberration frequency per cell for each aberration type o The frequency of aberrant cells for each aberration type.  Statistical analysis Several statistical analysis carried out based on: o Suggested number of cells analyzed o Number of animals per group o Number and range of doses o Aberration frequency for the test compound groups o Dose-response characteristics o The distribution of aberrations per cell and per animal Human Karyotype analysis Human Chromosomes preparation  Karyotype It is New Latin from Ancient Greek karyon, "kernel", "seed", or "nucleus", and typos, "general form". Mazen Zaharna Molecular Biology 1/2009 karyotype is the number and appearance of chromosomes in the nucleus of a eukaryotic cell. The term is also used for the complete set of chromosomes in a species or in an individual organism and for a test that detects this complement or measures the number. Mazen Zaharna Molecular Biology 1/2009 Karyotype is a test to: Identify and evaluate the size, shape, and number of chromosomes in a sample of body cells. Extra or missing chromosomes, or abnormal positions of chromosome pieces, can cause problems with a person's growth, development, and body functions. Mazen Zaharna Molecular Biology 1/2009  Chromosome Morphology Chromosomes are not visible under the light microscope in non-dividing cells (interphase cells). As the cell begins to divide, the threads of chromatin (DNA-protein complex) in the nucleus begin to condense into multiple levels of coiled structures recognizable as chromosomes. There are two modes of cell division: Mitosis: is responsible for the proliferation of body (somatic) cells, Meiosis: is responsible for the production of gametes. Because mitotic cells are easy to obtain, morphological studies are generally based on mitotic metaphase chromosomes.  Chromosome Analysis The best mitotic stage for chromosome analysis is metaphase. A typical metaphase chromosome consists of two arms separated by a primary constriction or centromere. Each of the two sister-chromatids contains a highly coiled double helix of DNA. Often the sister chromatids are so close to each other that the whole chromosome appears as a single rod-like structure A chromosome may be characterized by its total length and the position of its centromere.  Metaphase At metaphase the chromosomes are at their most condensed state. Spindle fibers attaching to the area of the centromere called the kinetochore, forming pole-chromosome fibers.  Chromosome Number The diploid chromosome number is the number of chromosomes in the somatic cell and is designated by the symbol 2N. The gametes, which have one half the diploid number, have the haploid number N. Thus, there are 23 pairs of chromosomes in human cells.  Of these, 22 pairs are not directly involved in sex determination, and are known as autosomes. The remaining chromosome pair consists of the sex chromosomes, and is directly involved in sex determination. In females the two sex chromosomes are identical (XX), whereas in males the two sex chromosomes are not identical (XY).  Karyotyping is done to:  Find out whether the chromosomes of an adult have a change that can be passed on to a child.  Find out whether a chromosome defect is preventing a woman from becoming pregnant or is causing miscarriages.  Find out whether a chromosome defect is present in a fetus.  Karyotyping also may be done to find out whether chromosomal problems may have caused a fetus to be stillborn.  Identify the sex of a person by checking for the presence of the Y chromosome. This may be done when a newborn's sex is not clear. Mazen Zaharna Molecular Biology 1/2009  Types of Tissue A variety of tissue types can be used to obtain chromosome preparations. Some examples include peripheral blood and bone marrow. In the case of blood cell culture only cells that are actively dividing can be used for cytogenetic studies. Normally only white blood cells are used for cytogenetic analysis. Specific techniques differ according to the type of tissue used. Practical work Mazen Zaharna Molecular Biology 1/2009  Overview of Procedure 1. Collection of blood 2. Cell culture 3. Harvesting: stopping the cell division at metaphase 4. Hypotonic treatment of red & white blood cells 5. Fixation 6. Slide preparation 7. Staining  1- Collection of blood Draw 5 ml of venous blood into a sterile heparinized tube containing 0.1 ml of sodium heparin (500 units/ml).  2- Cell Culture Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment under controlled conditions. Mazen Zaharna Molecular Biology 1/2009 but artificial environments consist of a suitable vessel with substrate or medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and gases (CO2, O2), and regulates the physio-chemical environment (pH buffer, osmotic pressure, temperature) Mazen Zaharna Molecular Biology 1/2009  2- Cell Culture Sterile technique must be used throughout the cell culture preparation, because it is possible to cause major contamination during this procedure. 70% of the problems are due to a lack of good sterile technique. Antibiotics do not eliminate problems of gross contamination which result from poor sterile technique or antibiotic-resistant mutants. Autoclaving renders pipettes, glassware, and solutions sterile.  Contamination of cell cultures Cell cultures can be contaminated by fungi, bacteria and viruses, These organisms can appear in cultures due to several factors: Through dust particles carried by air currents. Aerosols produced by the operator during handling. Through non-sterile equipment. Mazen Zaharna Molecular Biology 1/2009 Vertical Laminar Flow Horizontal Laminar Flow Mazen Zaharna Molecular Biology 1/2009 1-Preparation of complete culture medium Medium Pipette 4.5 ml RPMI 1640 (Roswell Park Memorial Institute) medium with L-Glutamine into a 15 ml labeled sterile culture tube.  2-Treatment and cultivation: Incubation Add 0.5 ml of whole heparinized blood into the tube containing the supplemented medium. Mix contents of tube with gentle inversion. Incubate in 5% CO2 incubator at 37oC for 48 hours. 3-Harvesting:  Harvesting: mitotic spindle formation is blocked: usually by adding colcemide (colchicine) to the culture, and the cell division is stopped at the metaphase level.  Colchicine solution was added by 0.05 ml to the culture tube after 46.5 hour.  Colchicine inhibits microtubule polymerization by binding to tubulin, one of the main constituents of microtubules 4- Hypotonic treatment of red & white blood cells  Centrifuge for 10 minutes at 1000 rpm.  Discard supernatant without disturbing the cells leaving 0.5 ml of fluid.  Add 8 ml of warm hypotonic solution (0.075 M o KCl) at 37 C.  Incubate at 37oC incubator for 20 minutes. Hypotonic solution should not be in contact with cells more than 27 minutes (may cause rupture of WBCs). 5- Fixation:  0.5 ml of cold Carnoy’s fixative solution were added to each tube and pipette gently break up cell’s clumps.  Centrifuge for 10 minutes at 1000 rpm.  Remove supernatant leaving about 0.5 ml of fluid on top of cells. At this time there is probably a small whitish or reddish film at the bottom of the tube, The film contain red blood cell debris and enlarged WBCs.  Add 0.05 ml of fixative to the tube.  Mix with a Pasteur pipette 3-4 times.  Centrifuge the tube for 10 minutes at 1000 rpm.  Remove supernatant and add another 0.2 ml of cold fixative, & mix well.  Centrifuge the tube for 10 minutes at 1000 rpm.  Repeat the last two steps.  Remove the supernatant leaving 1 ml of fluid at the bottom. 6- Slides Preparation:  The slide must be exceptionally clean.  Lay slides on a paper towel.  Withdraw a few drops of cell suspension into a pipette.  From a height of 20 cm, drop 2 or 3 drops of fluid on each slide.  Allow the slides to dry.  7- Staining Stain the slides by immersion in fresh Giemsa stain for 7-10 minutes Remove slides from stain & rinse in distilled water. Observe under microscope 40X then under oil immersion.  Human Numerical Disorders 1. Normal Karyotype (XX, XY) 2. Klienfilter syndrome (XXY) 3. Down Syndrome (Trisomy 21) 4. Turner Syndrome (Monosomy X) 5. Edward’s Syndrome (Trisomy 18) 6. Patau Syndrome (Trisomy 13) Mazen Zaharna Molecular Biology 1/2009 Thank You  Mazen Zaharna Molecular Biology 1/2009 X-Y Linked Inheritance  What are the sex chromosomes? A gene that is located on either sex chromosome is called a sex-linked gene Genes on the Y chromosome are called Y-linked genes; there are few of these Genes on the X chromosome are called X-linked genes What are the sex chromosomes? Genes are found in structures in our cells called chromosomes. The sex chromosomes, called the X and Y chromosome, determine our biological sex. Males inherit their X chromosome from their mothers and their Y chromosome from their fathers. Females inherit one X chromosome from each parent. Mutations or pathogenic variants in genes of these chromosomes can lead to symptoms of a genetic disease.  At Fertilisation When an X chromosome meets a Y chromosome at fertilisation, each sex-linked gene on the X chromosome becomes expressed in the phenotype of the human male produced. This is because his Y chromosome does not possess alleles of any of these sex-linked genes and cannot offer dominance to them. 4  Females have two X chromosomes, so they have a “back-up” second X chromosome. So, its less likely to have symptoms of an X-linked genetic disease than a male, and the symptoms are usually milder. she has a 25% chance to have a son who inherits Sex-linked the mutation and is likely to show symptoms of the genes X-linked disease. male is more likely to have symptoms of an X-linked genetic disease than a female. Males have a single X chromosome, with no “back-up” second X chromosome. When a male with an X-linked gene mutation has children All his daughters would inherit his mutation and be “carriers” None of his sons would inherit his mutation since his 5 sons get his Y chromosome  we challenge you to remember syndromes with an X-linked Recessive (XLR) inheritance pattern with the following mnemonic: CHAD’S KINKY WIFE GOT LUCKY - Chronic granulomatous disease - Hunter’s disease - Anhidrotic ectodermal dysplasia - Dyskeratosis congenita - SCID - Menkes Kinky hair disease - Wiskott-Aldrich - Ichthyosis - X-linked Fabry’s disease - Ehlers-Danlos V and IX - G6PD deficiency - Lesch-Nyhan  Chronic granulomatous disease The disease was first described in the 1950s as “a fatal granulomatosus of childhood“ a genetic disorder in which white blood cells called phagocytes are unable to kill certain types of bacteria and fungi More than half of cases of chronic granulomatous disease are transmitted genetically as an X-linked recessive trait. CGD occurs only in males Severe combined immunodeficiency (SCID) Severe combined immunodeficiency (SCID) is a group of rare disorders caused by mutations in different genes involved in the development and function of infection-fighting immune cells. Infants with SCID appear healthy at birth but are highly susceptible to severe infections. Menkes Kinky hair disease Menkes syndrome is a disorder that affects copper levels in the body. It is characterized by sparse, kinky hair; failure to gain weight and grow at the expected rate (failure to thrive); and deterioration of the nervous system. Wiskott-Aldrich Wiskott-Aldrich syndrome is a rare genetic immunodeficiency that keeps a child's immune system from functioning properly. It also makes it difficult for a child's bone marrow to produce platelets, making a child prone to bleeding. It occurs mostly in males.  G6PD deficiency Glucose-6phosphate dehydrogenase deficiency is a genetic disorder that affects red blood cells, which carry oxygen from the lungs to tissues throughout the body. In affected individuals, a defect in an enzyme called glucose-6-phosphate dehydrogenase causes red blood cells to break down prematurely.  Genetic Testing Predictive testing: Tells a person if she carries a mutation that will cause, or put her at higher risk for, a disease later in life. ? ? ? ? Newborn screening: Detects common disorders in newborns, where immediate treatment can prevent dangerous symptoms Carrier testing : Tells a person whether or not he carries a mutation that could be passed on to his offspring. One can be a carrier, but not be at risk for a disease (as in recessive genes)  x23 x23 x23 x23 x23 x23 x23 x23 x23 x23 x23 x23 x23 x23 x23 x23 x23 x23 x23 x23 Genetic testing DNA RNA Protein Function  How do we “look” at the DNA sequence? Sequencing PCR RFLP analysis Gel Probes electrophoresis Southern blot Dot blot Microarray Benefits of Genetic Testing If Negative If Positive Relief Able to make informed decisions Fewer health check-ups and May be possible to reduce the tests that go with being in a risk of coming down with severe family that tends to have a symptoms higher risk of a particular genetic diseases Limitations of Genetic Testing Mutations may not always lead to disease Existing tests only look for the most common mutations, some disease causing mutations will not be detected by conventional tests Small chance of errors in testing procedure Testing is not always matched by treatment Puzzle 1 18 Puzzle 1 - Answer 19 Puzzle 2 20 Puzzle 2 - Answer 21  Y-linked Inheritance Y-linked traits are controlled by alleles on the Y chromosome Another word for Y-linked traits is holandric traits as they are “wholly male”.  Genetic Diseases, Y-Linked diseases: Hairy Ears Pseudoautosomal Linkage of Hodgkin Disease Retinitis Pigmentosa, Y-Linked Sertoli cell-only syndrome, Y-linked Spermatogenic Failure, Nonobstructive, Y-Linked Hypertrichosis, or "Hairy Ears"  Hodgkin Disease Hodgkin's lymphoma is a type of cancer that affects the lymphatic system, which is part of the body's germ-fighting immune system. In Hodgkin's lymphoma, white blood cells called lymphocytes grow out of control, causing swollen lymph nodes and growths throughout the body.  Retinitis pigmentosa Retinitis pigmentosa is a group of related eye disorders that cause progressive vision loss. These disorders affect the retina, which is the layer of light-sensitive tissue at the back of the eye. In people with retinitis pigmentosa, vision loss occurs as the light-sensing cells of the retina gradually deteriorate.  Sertoli cell-only syndrome, Y-linked Sertoli cell-only syndrome, also known as del Castillo syndrome or germ cell aplasia, is one of the most common causes of azoospermia in infertile men. In this syndrome, only Sertoli cells line the seminiferous tubules of the testes, and the patients have very low or absent spermatogenesis  Spermatogenic Failure, Nonobstructive, Y-Linked Y chromosome infertility is a condition that affects the production of sperm and causes male infertility  Assignment Write down a method to examine the Barr body in the following conditions: 1. Normal female 2. Triple X syndrome 3. Turner syndrome 4. Normal male Numerical Aberrations  Numerical Aberration Any change in chromosomal number leading to disturbance of naturally occurring balance I.e., Wild type of chromosomes Numerical Aberrations Aneuploidy Polyploidy Monosomy Trisomy Definitions Aneuploidy: It is the change in the number of parts of chromosome sets involving one or 2 chromosomes. Monosomy: a condition in which only one chromosome is missing out of 46 chromosomes and karyotype is 45 in case of human. Trisomy: a condition in which an organism or a cell has one chromosome more than the normal somatic complement of the species. Polyploidy: it’s the multiplication of the whole set of chromosome Down Syndrome Aneuploidy Down’s syndrome Case: (Trisomy 21 ) Karyotype: (47, XX/XY, +21) Symptoms: Typically there are epicanthal folds and a flat, broad face. Mental and Physical Retardation Delays in speech & attention problems. Protruding tongue. Hypotonia (poor muscle tone) Aneuploidy Down’s syndrome Aneuploidy Down’s syndrome Defect Gene Gene Function Studies suggest that in the brain, it helps direct the App movement (migration) of nerve cells (neurons) during early development. Neurodegenerative disorders In nerve cells (neurons), the DYRK1A enzyme is involved in the formation and maturation of dendritic dyrk1a spines from dendrites. Dendrites are specialized extensions from neurons that are essential for the transmission of nerve impulses. Regulates the intracellular concentration of folate which Cardiac defects SLC19A1 improves the endothelial function of cardiovascular tissue This gene encodes a transcription factor which regulates genes involved in development and Leukaemia ets2 apoptosis. The encoded protein is also a proto- oncogene and shown to be involved in regulation of telomerase. Patau Syndrome Aneuploidy Patau syndrome Case: (Trisomy 13 ) Karyotype: (47, XX/XY, +13) Symptoms: cleft lip and palate an abnormally small eye or eyes (microphthalmia) smaller than normal head size (microcephaly) skin missing from the scalp (cutis aplasia) ear malformations and deafness Polydactyly and closed fist Edward Syndrome Aneuploidy Edward Syndrome Case: (Trisomy 18 ) Karyotype: (47, XX/XY, +18) Symptoms: low birth weight small head and jaw an unusual-looking face and head unusual hands and feet with overlapping fingers and webbed toes problems with feeding, breathing, seeing and hearing Kleinfelter Syndrome Aneuploidy in Sex Chromosomes: Klinefelter Syndrome Case: (trisomy 23) Karyotype: (47, XXY, +X) Restricted to Males Aneuploidy in Sex Chromosomes: Klinefelter Syndrome Symptoms: in babies and toddlers – learning to sit up, crawl, walk and talk later than usual, being quieter and more passive than usual in childhood – shyness and low self-confidence, problems with reading, writing, spelling and paying attention, mild dyslexia or dyspraxia, low energy levels, difficulty socialising or expressing feelings in teenagers: growing taller than expected for the family (with long arms and legs), broad hips, poor muscle tone and slower than usual muscle growth, reduced facial and body hair that starts growing later than usual a small penis and testicles, and enlarged breasts (gynaecomastia) in adulthood – inability to have children naturally (infertility) Aneuploidy in Sex Chromosomes: Klinefelter Syndrome Jacob's Syndrome Aneuploidy in Sex Chromosomes: Jacobs syndrome Case: (trisomy 23) Karyotype: (47, XYY, +Y) Restricted to Males Characteristics of XYY syndrome are often subtle and do not necessarily suggest a serious chromosomal disorder. Thus, males with this condition are often undiagnosed or misdiagnosed. Aneuploidy in Sex Chromosomes: Jacob Syndrome Increased height, becomes apparent after the age of five or six. Some individuals with XYY also develop severe cystic acne during adolescence. Fertility and sexual development are normal. Normal physical appearance (phenotype). Boys with XYY syndrome typically have normal intelligence, Although, on average, IQ is 10 to 15 points lower than siblings. Learning disabilities have been reported in up to 50 percent of cases, most commonly speech delays and language problems. Reading difficulties are common due to an increased incidence of dyslexia. behavioral problems such as an explosive temper, hyperactivity, impulsivity, defiant actions, or, in some cases, antisocial behavior. Triple X Syndrome Aneuploidy in Sex Chromosomes: Triple X syndrome Case: (trisomy X) Karyotype: (47, XXX, +X) Restricted to Females Occasionally, significant symptoms may occur, which vary among individuals. As the previously mentioned syndromes, females with triple x syndrome suffer from Behavioral problems, Learning disabilities and Psychological problems, such as anxiety and depression  Aneuploidy in Sex Chromosomes: Triple X Syndrome Symptoms: Vertical folds of skin that cover the inner corners of the eyes (epicanthal folds) Widely spaced eyes Curved pinky fingers Flat feet Breastbone with an inward bowed shape Weak muscle tone (hypotonia) Seizures Ovaries that don't work properly at a young age (premature ovarian failure) Turner Syndrome Aneuploidy in Sex Chromosomes: Turner Syndrome Case: (Monosomy X) Karyotype: (45, X0, -X) Restricted to Females In females who have Turner syndrome, one copy of the X chromosome is missing, partially missing or changed.  Aneuploidy in Sex Chromosomes: Turner Syndrome Symptoms: particularly short, wide neck (webbed neck) a broad chest and widely spaced nipples arms that turn out slightly at the elbows a low hairline and teeth problems many moles eyes that slant downwards droopy eyelids (ptosis) Underdeveloped gonads Aneuploidy in Sex Chromosomes: Turner Syndrome Genetic Causes in Turner Syndrome About half of individuals with Turner syndrome have monosomy X, Turner syndrome can also occur if one of the sex chromosomes is partially missing or rearranged rather than completely absent. Some women with Turner syndrome have a chromosomal change in only some of their cells, which is known as mosaicism. Women with Turner syndrome caused by X chromosome mosaicism are said to have mosaic Turner syndrome. Lab Activity Aim: Observation of different stages of meiosis under microscope 1. Flower buds of appropriate sizes were collected from palnts and fixed in Carnoy’s fluid for 24 hrs. 2. Buds are then transferred to 70% ethanol and stored at 40°C 3. Flower buds are then transferred individually on a clean glass slide 4. A single anther is dug up from flower bud 5. A small drop of 1% acetocarmine stain was placed over the anther and the latter was gently tapped with flat spatula to release pollen mother cells into the stain 6. The anther lobe wall/ debris was carefully removed from the stain before placing on to it the cover slip 7. The preparation was then gently squashed using vertical thumb pressure applied on to the coverslip under double fold of filter paper 8. Observe under light microscope Assignment As we discussed in the previous weeks several abnormalities, Monosomy 7 and Monosomy 11 are mentioned as Mosaic abnormalities. Define Mosaic Abnormalities? Identify the Type of this mutation (Germline / Somatic) ? Mention the disease related ? Structural Chromosomal Aberrations Chromosomal Aberrations Any kind of alteration in the genetic information (Chromosomes) can have a deleterious effect on the organism. These alterations can be the manifestation of variations in the number of chromosomes (discussed in another chapter) or alterations in the chromosomal structure.  Background Structural Aberrations Numerical (Aneuploidy) Change to number of chromosome Structural Change to a specific part of the chromosome Deletion Inversion Structural Aberrations Duplication Translocation Polyploidy Aneuploidy Inversion  Inversion Chromosome inversions are defined as the rearrangement produced by two break - points within the same chromosome, with the subsequent inversion and reinsertion of this fragment.  Inversion if the inverted fragment Pericentric includes the chromosome’s centromere Chromosome inversions may be if the inverted fragment Paracentric does not include the chromosome’s centromere. The frequency in the general population of chromosome inversions is 1– 5 in 10,000 for paracentric inversions and 1–7 in 10,000 for pericentric inversions.  Inversion In most cases, being a carrier of a chromosome inversion has no direct health implications. However, carriers of a balanced Robertsonian translocation have some reproduction implications, due to imbalanced gametes: Offspring with an unbalanced chromosome Reproduction issues: rearrangement: Sterility/infertility;​ In unbalanced pericentric inversions: formation of Recurrent pregnancy losses (in 3%– recombinants with deletion and duplication of the 9% of couples with recurrent inverted segment; pregnancy losses).​ In unbalanced paracentric inversions: inverted duplications of the segments Deletion  Deletion In deletion the daughter chromosome loses a part of chromosome compared to parent chromosome, and this deleted part of chromosome is degraded and completely lost, which makes this kind of mutation irreversible Human disorders caused by deletion of large segment of chromosome are usually the cases in which deletion occurs in heterozygous individuals as the deletion in homozygous would result in lethal condition.  Deletion Types of deletion: Terminal Deletion - a deletion that occurs towards the end of a chromosome. Intercalary Deletion - a deletion that occurs from the interior of a chromosome.  Deletion Terminal Deletion :A single break at the terminal portion of chromosome causes the loss of genetic material. Example: 1- Wolf-Hirschhorn syndrome 2- Cri-du Chat Syndrome Wolf-Hirschhorn syndrome Deletion of genetic material near the end of the short (p) arm of chromosome 4. NSD2, LETM1, and MSX1 are the genes that are deleted in people with the typical signs and symptoms of this disorder. These genes play significant roles in early development. Symptoms: 1- Greek helmet face 2- Mental Retardation 3- Frequent Seizures 4- Microcephaly 5- Cardiac, renal and genital abnormalities Wolf-Hirschhorn syndrome NSD2 gene is associated with many of the characteristic features of Wolf-Hirschhorn syndrome, including the distinctive facial appearance and developmental delay. Deletion of the LETM1 gene appears to be associated with seizures or other abnormal electrical activity in the brain. The loss of the MSX1 gene may be responsible for the dental abnormalities and cleft lip and/or palate that are often seen with this condition.​ Cri-du chat syndrome Cri-du-chat syndrome is caused by a deletion of the end of the short (p) arm of chromosome 5 (5p-) Researchers believe that the loss of a specific gene, CTNND2, is associated with severe intellectual disability in some people with this condition. Symptoms: 1-A high-pitched, cat-like cry or weak cry 2-low birth weight 3-small head 4-broad, flattened bridge of the nose 5-eyes spaced wide apart 6-folds of skin over the eyelids Cri-du chat syndrome The CTNND2 gene provides instructions for making a protein called delta -catenin. This protein is active in the nervous system, where it likely helps cells stick together (cell adhesion) and plays a role in cell movement. In the developing brain, it may help guide nerve cells to their proper positions as part of a process known as neuronal migration.  Deletion Intercalary Deletion: An intermediate segment of the chromosome is lost, leaving the ends of the chromosome intact. This is the result of two breaks in the chromosome followed by the union of the two ends Example: 1- Prader-Willi syndrome (PWS) and Angelman syndrome (AS) 2-William's disease Prader-Willi syndrome Multisystem, contiguous gene disorder caused by an absence of paternally expressed genes within the 15q11.2-q13 region via deletion of the paternally inherited. Symptoms: (Infants) 1- Poor muscle tone 2- Underdeveloped genitals. 3- Almond-shaped eyes 4- Narrowing of the head at the temples 5- Turned-down mouth and a thin upper lip 4- Generally poor responsiveness  If the previously mentioned mutation occurred on the maternal chromosome. It results in another syndrome Angelman syndrome (AS) Angelman syndrome (AS) is a rare neuro -genetic disorder that occurs in one in 15,000 live births or 500,000 people worldwide. (deletion in chromosome 15) It is caused by a loss of function of the UBE3A gene in the 15th chromosome derived from the mother. Symptoms: 1- Being restless (hyperactive) 2- Having a short attention span. 3- A wide mouth with widely spaced teeth 4- Tendency to stick the tongue out 5- A side-to-side curvature of the spine Angelman syndrome (AS) UBE3A, the gene that encodes E6AP, is a protein that is expressed in an imprinted manner in the brain. Genomic imprinting marks the parental origin of chromosomal subregions and results in allele-specific differences in DNA methylation, transcription, and replication. Within the chromosome region 15q11-q13, the gene UBE3A is imprinted specifically in the brain, resulting in maternal expression of E6AP in the human fetal brain and adult cortex, while the paternal copy is silenced.  William's syndrome Williams Syndrome is caused by microdeletion at 7q11.23. This condition is characterized by mild to moderate intellectual disability or learning problems, unique personality characteristics, distinctive facial features, and heart and blood vessel (cardiovascular) problems. Symptoms: (Infants) 1- Chronic ear infections and/or hearing loss. 2- Dental abnormalities, such as poor enamel and small or missing teeth. 3- Endocrine abnormalities: hypothyroidism, early puberty and diabetes in adulthood. 4- Scoliosis (curve of the spine). 5- Unsteady walk (gait). Translocation Translocation It is the shuffling of genetic material along the length of chromosomes. The inter-chromosomal translocation involving two nonhomologous chromosomes, one donor and the other recipient chromosome 20 There are 2 types of translocation: 20 a- Reciprocal Translocation b- Robertsonian Translocation Translocation Reciprocal Translocation Reciprocal translocations are the transfer of genetic material between homologous chromosomes. These are the most balanced exchanges, such that no genetic material is lost, and individuals are phenotypically normal. A prototypical example of this phenomenon is represented by the Philadelphia chromosome associated with acute lymphocytic leukemia and chronic myelogenous leukemia Translocation Example 1: Philadelphia chromosome. A piece of chromosome 9 and a piece of chromosome 22 break off and trade places. The BCR-ABL gene is formed on chromosome 22 where the piece of chromosome 9 attaches. The changed chromosome 22 is called the Philadelphia chromosome. This translocation results in a fusion gene called the BCR-ABL gene. Translocation Example 1: Philadelphia chromosome. ABL This gene is a protooncogene that encodes a protein tyrosine kinase involved in a variety of cellular processes, including cell division, adhesion, differentiation, and response to stress. The activity of the protein is negatively regulated by its SH3 domain, whereby deletion of the region encoding this domain results in an oncogene. BCR-ABL Fusion gene BCR-ABL1 oncogene that encodes the chimeric BCR-ABL1 protein with constitutive strong tyrosine kinase activity and this activity is the significant molecular biological basis for the pathogenesis of these diseases. Translocation Robertsonian Translocation Robertsonian translocations (RobT) result from the breakage of two acrocentric chromosomes (13, 14, 15, 21, and 22) and subsequent fusion of their long arms to form one derivative chromosome. The short arms are lost, and the total chromosome number is reduced to 45. Genetically balanced carriers of these translocations have an increased incidence of infertility as well as a risk for genetic imbalances among their offspring. The risk of Down syndrome (trisomy 21) and Patau syndrome (trisomy 13) is elevated in the offspring of the rob(14;21) and the rob(13;14) balanced carriers Translocation Robertsonian Translocation & Down Syndrome Approximately 4% of Down syndrome patients have 46 chromosomes, one of which is a Robertsonian translocation between chromosome 21q and the long arm of one of the other acrocentric chromosomes (usually chromosome 14 or 22). The translocation chromosome replaces one of the normal acrocentric chromosomes, and the karyotype of a Down syndrome patient with a Robertsonian translocation between chromosomes 14 and 21 is therefore 46,XX or XY,rob(14;21)(q10;q10),+21 Translocation Robertsonian Translocation & Down Syndrome Despite having 46 chromosomes, patients with a Robertsonian translocation involving chromosome 21 are trismic for genes on the entirety of 21q Duplication Repetition of a segment of chromosome containing several genes. Depending upon the location and orientation of the duplicated segment, the duplication can be divided into several categories The Most important are: 1- Tandem Duplication 2- Non-Tandem Duplication Examples: 1- Charcot-Marie-Tooth disease type 1A Duplication Example 1: Charcot-Marie-Tooth disease type 1A This is a type of inherited neurological disorder that affects the peripheral nerves. CMT1A is caused by having an extra copy (a duplication) of the PMP22 gene. PMP22 The PMP22 gene provides instructions for making a protein called peripheral myelin protein 22 (PMP22). This protein is found in the peripheral nervous system, which connects the brain and spinal cord to muscles and to sensory cells that detect sensations such as touch, pain, heat, and sound. PMP22 PMP22 plays a crucial role in the development and maintenance of myelin. Studies suggest that the PMP22 protein is particularly important in protecting nerves from physical pressure, helping them restore their structure after being pinched or squeezed (compressed). The PMP22 gene also plays a role in the growth of Schwann cells and the process by which cells mature to carry out specific functions (differentiation).  Fluorescence in situ hybridization (FISH) What is FISH ? It is process of painting the whole chromosome or only part of the chromosomes (protein) with florescence molecule to identify chromosomal abnormality and presence of a region of DNA or RNA within the chromosome. History: ⚫In 1969, Gall and Pardue introduced in situ hybridization (ISH) to localize nucleic acids in individual cells. ⚫Pinkel (1986), described the Immunofluorescence technique. ⚫Afterwards, so many techniques derived from FISH have been developed. Other techniques: ⚫Q-FISH. ⚫Fiber FISH. ⚫Multi colour FISH(M-FISH or SKY). ⚫Flow-FISH. ⚫Comparative genomic hybridization(CGH). ⚫SNP(single nucleotide polymorphism array). Targets: ⚫ Metaphase chromosomes. ⚫ Interphase cells. ⚫ Tissue sections---from tissue biopsy slides. ⚫ Cells in culture. What does FISH need? ⚫Sample. ⚫Probe. ⚫Fluorescence microscope. Samples: ⚫ Blood. ⚫ Bone marrow(aspirate , biopsy).  Probe: It is a specific DNA fragment, usually 1 to 100 kb length, complementary to the chromosome site that we are interested in e.g of probe:  Single colour  Dual colour _ dual fusion  Dual colour _ single fusion Probe Labeling: Direct labeling : the probedirectly labeled with fluorochromes such as Spectral Green and Spectral Orange(One-step hybridization). Indirect labeling : needs antibodies to complete FISH procedure( Haptens-Biotin-dUTP, digoxigenin-dUTP). Fluorescence microscope: Light source: High-pressure mercury vapor lamps, tungsten-halogen lamps or xenon lamps. Filters: 1. Exciting filter: to let a certain wave length of light passes so that can excite the given fluorochrome carried on sample. 2. Barrier filter: to allow the visible light passes so that the f luorescence can be seen by eyes or the image can be captured. Note: there should be no auto-fluorescence in any part of light path except for samples Procedure: Denaturation of chromosome. Denaturation of probe. Hybridization: It is the formation of duplex between 2 complementary sequences. Florescence staining. Examination of the slide or store in dark.  The principles of fluorescence in situ hybridization: (a) The basic elements are a DNA probe and a target sequence. (B) Before hybridization, the DNA probe is labelled indirectly with a hapten (left panel) or directly labelled via the incorporation of a fluorophore (right panel). (C)The labelled probe and the target DNA are denatured to yield single- stranded DNA. (D)They are then combined, which allows the annealing of complementary DNA sequences. (E)If the probe has been labelled indirectly, an extrastep is required for visualization of the non-fluorescent hapten that uses an enzymatic or immunological detection system. Finally, the signals are evaluated by fluorescence microscopy A good FISH method should have: ⚫ An extremely high specificity(extremely low background). ⚫ A good sensitivity(good hybridization efficiency). ⚫ Unambiguous recognition of the hybridization signal. FISH limitation: Probe design requires knowledge of specific chromosomal abnormality to be studied. Cut off signals may be different among laboratory. Processing error, imperfect hybridization, non specific binding, photo bleaching, inter observer variability and false positive and negative. Advantages: ⚫ It is useful in establishing the percentage of neoplastic cells at the time of diagnosis and after therapy. ⚫ FISH studies are used to investigate the origin and progression of hematologic malignancies and to establish which hematopoietic compartments are involved in neoplastic processes. ⚫ Quick and correct results save time and money by preventing unnecessary additional diagnostics and suboptimal treatment approaches. ⚫ less labor-intensive method for confirming the presence of a DNA segment within an entire genome than other conventional methods Advantages: ⚫ Fast 48-72 hr. ⚫ Reliable and unbiased result. ⚫ Architecture of specimen remain intact. ⚫ Old specimen can be used up to (7 years). ⚫ It can detect numerous abnormalities( gain, losses of whole chromosome or deletion/duplication) superior to PCR. ⚫ Used in diagnostic evaluation specially like cryptic abnormality that not evident by conventional karryotyping. Application of FISH in haematology: ⚫ Acute Lymphoblastic Leukemia t(4;11). ⚫ Acute Myeloid Leukemia( PML/RARA t(15;17), AML/ETO t(8;21) ⚫ Chronic Myeloid Leukemia( ABL/BCR t(9;22). ⚫ Chronic Lymphocytic Leukemia(del13q14). ⚫ Myelodysplastic Syndromes. ⚫ Plasmacell myeloma( deletion of 13q 14 or monosomy 13). ⚫ Non Hodgkin Lymphoma(t(11;14) in mantle cell lymphoma). ⚫ CLL/small lymphocytic lymphoma. ⚫ Mismatched BM transplantation diseases relapse.