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• Human Cytogenetics


  A branch of genetics that studies the organization of human chromosomes, and their number, structure, function, behavior in relation to gene inheritance, and expression for identification of genomic mutations that cause diseases
• Karyotyping


  The process of pairing and ordering all metaphase chromosomes of an organism in a standard fashion in order for providing a genome-wide photograph of an individual's chromosomes

  
• FISH technique


  Fluorescence in situ hybridization, a molecular-cytogenetic testing method that uses fluorescent probes to evaluate loss or duplication of genes and/or DNA sequences
• Cytogenetics involves testing samples of tissue, blood, or bone marrow in a laboratory to look for changes in chromosomes, including broken, missing, rearranged, or extra chromosomes
• Chromosome


  The highly condensed form of chromatin fibers in metaphase stage, which is formed from a complex of DNA and proteins

  
• Chromosome formation


  1. DNA and proteins are wrapped into nucleosomes

  2. solenoids

  3. chromatin fibers

  4. metaphase chromosomes

  
• Metaphase chromosome


  The traditional subject for cytogenetic analysis because they are contracted, thicker enough and take up stains to be visible and examined with light microscope

  
• Metaphase chromosome


  • p arm - the smaller of the two arms, p stands for petite
  • q arm - the longer of the two arms
  • Bands are numbered from centromere outward

  
• Human diploid cells


  Normally have 23 pairs, or 46 chromosomes, contain two copies of each one of autosomes and two sex chromosomes, either two X chromosomes or an X and a Y chromosome

  
• Human haploid cells


  Eggs and sperm cells have 23 chromosomes, contain only one copy of each chromosome
• Types of chromosomes


  • Autosomes
  • Sex chromosomes
• Autosomes


  All chromosomes in humans other than the sex chromosomes, numbered 1-22 by size (starting with the largest to the smallest), homologous pairs because similarity in size and shape and type and loci of genes

  
• Sex chromosomes


  The X and Y chromosomes involved in sex determination, have different sizes and shapes
• Non-hereditary alterations in the number and structure of chromosomes can cause chromosome disorders in growth, development, function, and medical problems with body's systems
• Causes of chromosome abnormalities


  • Cell division error during the formation of reproductive cells
  • Cell division error after zygote formation, during early fetal development
  • Cell division error during person's life (Acquired after birth)
  • Some structural disorders inherited from parents
• Factors relative to chromosome abnormalities


  • Errors in Meiosis (increased maternal age, exposure to environmental mutagens)
  • Errors in Mitosis (exposure to environmental mutagens)
• Meiosis error


  1. Numerical abnormality occurs when chromosomes don't split into equal halves (nondisjunction) resulting in an egg or sperm with extra chromosomes, or missing chromosomes

  2. Structural abnormality occurs due to abnormal rearrangements during crossing over
• Pregnancy with a trisomic, monosomic embryo or with structural chromosome abnormalities may go to full-term and result in the birth of a child with health problems, may miscarry in the early stages, or may result in a baby born without signs of life (stillbirth)
• Mitosis error


  1. Chromosomes do not split into equal halves (nondisjunction), the new cells can have an extra chromosome or have a missing chromosome (Genetic Mosaicism)

  2. Errors in mitosis are responsible for some cases of mosaicism when chromosomal abnormalities occur in certain cells of the body but the other cells are with normal number of chromosomes

  3. Can occur in Somatic cell later after birth result in chromosome abnormalities and cancers
• Maternal age


  Women are born with all eggs they will ever have, and they begin to mature during puberty. Chromosomal aneuploid oocytes ovulation occurs with greater frequency in women above 35-40 years of age. Errors in meiosis may be more prone to happen as a result of the aging process
• Environmental conditions, exposure to radiations, taking medications, and foods, toxins can be the reason for a baby to be born with chromosome abnormality
• Karyotyping is done to find out any chromosomal abnormalities of parents before passing on to a child, find out chromosomal abnormalities that lead to reproductive problems, find out chromosomal abnormalities present in abortuses, find out chromosomal abnormalities involved in a baby's birth defects or disability, help diagnose chronic myelogenous leukemia (CML) and determine the appropriate treatment for some types of cancer, and identify the sex of a person when a newborn's sex is not clear

  
• Karyotyping


  Can be used to detect a variety of genetic disorders by identifying the change in number of chromosomes and finding gross structural abnormalities such as large deletions, duplications or other abnormal rearrangements by G-banding staining

  
• Routine karyotyping


  Chromosomes are fixed, spread on microscope slides, partially digested by trypsin and stained. The distinct bands of each chromosome revealed by using differential staining technique to allow for analysis of the chromosomal structure and identifying them individually

  
• Biological samples needed for cytogenetic and molecular-genetic analysis


  • Peripheral blood (white blood cells – lymphocytes)
  • Cheek swab (epithelial cells)
  • Bone marrow biopsy
  • Tissue biopsy (tumor tissue)
  • Amniotic fluid
  • Placental villi (Chorionic villus biopsy)
• Overview of karyotyping procedure


  1. Draw 3 to 5 ml of blood

  2. Add a few drops of blood (T-lymphocytes) into a culture

  3. Add phytohemagglutinin to stimulate mitosis

  4. Incubate at 37°C for 2 to 3 days

  5. Add colchicine to culture for 1 to 2 hours to stop mitosis in metaphase

  6. Centrifuge to concentrate cells. Add low-salt solution to eliminate RBCs and make lymphocytes to swell and cell burst by dropping

  7. Transfer cells to tube containing fixative to preserve chromosomes morphology

  8. Suspended Cells are dropped on glass slides to spread chromosomes. After drying and treating by trypsin, slides are stained with Giemsa stain

  9. Examine with microscope

  10. Digitised chromosome images processed to make karyotype
• Chromosome groups


  • Group A (1-3)
  • Group B (4,5)
  • Group C (6-12,X)
  • Group D (13-15)
  • Group E (16-18)
  • Group F (19,20)
  • Group G (21,22,Y)
• Identifying chromosomes


  • Size
  • Banding pattern (Giemsa bands)
  • Centromere position
  • Presence of satellites

  
• 1. banding


  Giemsa stain gives chromosomes a striped appearance, with dark G-bands formed by regions of DNA that are rich with adenine (A) and thymine (T) base pairs (heterochromatic regions), and light G-bands formed by regions rich with guanine (G) and cytosine (C) (euchromatic regions)

  
• Description of karyotype of chromosome abnormalities


  • Numerical abnormalities (e.g. 47,XX,+21)
  • Structural abnormalities (e.g. 46,XY,dup(1)(q22q25))

  
• Fluorescence in situ hybridization (FISH)


  A molecular-cytogenetic testing method that uses fluorescent probes to evaluate loss or duplication of genes and/or DNA sequences
• Numerical abnormalities


  For example a female Down syndrome or trisomy 21 is written as 47,XX,+21
• Structural abnormalities


  Changes are designated by letters, for example 'dup' for duplication such as 46,XY,dup(1)(q22q25 ) (duplication of a segment in long arm of chromosome 1, q, in region 2 between bands 22 and 25)
• Karyotype of chromosome abnormalities


  • 47,XX,+21
  • 46,XY,dup(1)(q22q25 )

  
• Fluorescence in situ hybridisation (FISH)


  The use of a fluorescent DNA or RNA probe to detect complementary genetic material in cells or tissue. In situ hybridization involves hybridizing a nucleic acid labeled with fluorescent dye to suitably prepared cells or tissues on microscope slides to allow visualization in situ (in the normal location on chromosome).
• Fluorescence in situ hybridisation (FISH)


  • Evaluates loss or duplication of genes and/or DNA sequences at certain locus on interphase chromosomes
  • Detects micro structural aberrations in metaphase chromosomes
  • Detects aneuploidies (numerical chromosomal abnormalities) in interphase or metaphase chromosomes

  
• Fluorescent probes


  Short sections of single-stranded DNA labeled with fluorescent tag that are complementary to the specific portions of DNA of interest on chromosome
• Conditions where FISH analysis is indicated


  • Unsuccessful cultivation
  • When the tissue from patient is not possible to be cultivated (Preimplantation analysis, Rapid prenatal examinations, Examinations of solid tumors or autopsy material)
  • Analysis of complicated chromosomal rearrangements (inversion, microdeletion, microduplication, microtranslucation)
  • Screen the sex chromosome in an infant with ambiguous genitalia
• FISH Procedure


  1. Slides preparation from cultured (Metaphase) or uncultured (Interphase) cells

  2. Denature the probe

  3. Hybridization by exposing the nuclear genome to a probe

  4. Fluorescence staining

  5. Visualization (Examine slides)
• Visualization of the Probe


  DNA probe is labeled with a colored fluorescent molecule that emits a particular color when viewed through a fluorescence microscope