Cytogenetics: Chromosome Analysis

• Analysis of chromosomes occurs during interphase and mitosis

Cytogenetics

• Cytogenetics studies the organization of the genome on a chromosome level
• There are two ways to analyze chromosomes:
  • In dividing cells during metaphase (mitosis)
  • In nondividing cells during interphase
• Classical cytogenetics involves karyotyping based on Denver and Paris standards
• Molecular cytogenetics includes FISH (fluorescent in situ hybridization) and aCGH (array comparative genomic hybridization)

Cytogenetical Analysis in Metaphase (Mitosis)

• Condensed chromosomes are placed in the equatorial plain for metaphase analysis
• A metaphase chromosome consists of:
  • A p-arm
  • A q-arm
  • A centromere
  • Chromatids
• Sufficient number and quality of metaphase chromosomes is required, achieved by:
  • Cultivating cells (in vitro) to gain an adequate amount of dividing cells
  • Halting cell division in metaphase using mitotic poisons like colchicine and colcemid
  • Hypotonization for optimal spreading of chromosomes on the slide
  • Staining to visualize the chromosomes
• A karyotype can be constructed using this method

Classification of Chromosomes

• Substantial staining techniques are used for classification of chromosomes
• Diffusional (homogenous) staining with Giemsa solution is used in the Denver classification of chromosomes
• Identification staining is used in the Paris classification and includes G-, Q-, R-, and C-banding

Denver Classification

• Denver classification involves chromosomes stained by diffusional (homogenous) staining
• This staining does not allow the recognition of euchromatin or heterochromatin
• Evaluation criteria includes the relative length of the chromosome and the position of the centromere (length of arms)
• Types of chromosomes are:
  • Big, medium, and small
  • Metacentric, submetacentric, and acrocentric
  • Telocentric chromosomes do not exist in humans
• Evaluation includes the total number of chromosomes and their number in particular groups A-G:
  • Group A: Big metacentric chromosomes 1-3
  • Group B: Big submetacentric chromosomes 4-5
  • Group C: Medium submetacentric chromosomes 6-12 + chromosome X
  • Group D: Medium acrocentric chromosomes 13-15
  • Group E: Small metacentric and submetacentric chromosomes 16-18
  • Group F: Small metacentric chromosomes 19-20
  • Group G: Small acrocentric chromosomes 21-22 + chromosome Y
• Classical karyotypes do not allow individual chromosome identification within a group or reveal smaller chromosome reorganizations
• Normal male - 46,XY
• Normal female - 46,XX

Paris Classification

• Involves staining chromosomes with identification staining techniques after preliminary treatment with heat or digestive enzymes
• Results in banding of chromosomes, which responds to the euchromatin and heterochromatin structure of each chromosome
• Allows for identifying each chromosome in a group
• Allows for precise detection of rearrangement of chromosomes
• Each arm is divided into a certain number of regions and contains a certain number of bands, regarding the size of the arm
• Numbering of regions and bands starts from the centromere towards the telomeres
• Evaluation includes the count and position of bands on both arms of each chromosome
• Each band is described by the chromosome number, arm symbol, region number, and band number
• The record is writing and reading without interruption and punctuation (e.g., 7q22)

Staining Techniques

• Commonly used identification staining techniques include Q, G, R, and C – bandings
• Q-banding involves:
  • The first identification staining technique from 1969
  • Chromosomes treated with heat and quinacrine dihydrochloride mustard (fluorochrome) that preferentially binds to heterochromatin clusters
  • Bands that are more or less visible in UV light (in a fluorescent microscope)
• G-banding is:
  • A used technique in cytogenetics
  • Chromosomes are treated with trypsin (proteolytic enzyme) and then stained with Giemsa dye
  • Creates gained bands that are identical with Q-bands
• R-banding is:
  • Used for bands that are reverse to those of Q- and G-bands
• C-banding:
  • Demonstrates heterochromatin blocks at the centromeric region of all chromosomes and at pericentromeric regions of chromosomes 1, 9, 16, and Yq
• About 400 bands can be distinguished in analyzing metaphase chromosomes
• High-resolution techniques were prepared using prometaphase (prophase) = less condensed chromosomes to analyze euchromatine and heterochromatine segments in more detail
• Each sub-band is named by its number (behind the dot), for example 7q22.1
• The number of bands obtained increases to 1400
• The cytogenetic map location of the CFTR gene is 7q31.2
• There are 650 bands in HT-- 46,XY

Cytogenic Analysis in Interphase

• Chromatin is divided into two subtypes:
  • Euchromatin: Gene-rich, transcriptionally active, condensed only in the M-phase of the cell cycle
  • Heterochromatin: Gene-poor, transcriptionally silent, condensed during the whole cell cycle
• There are two types of heterochromatin:
  • Constitutive: Presents in all cells of the organism during all its development and life
    • Includes heterochromatin bands of chromosomes
    • Includes centromere regions of all chromosomes
    • Includes pericentromeric blocks of chromosomes 1, 9 and 16
    • Includes the part of longer (q) arms of the chromosome Y
  • Facultative: Depends on the developmental stage of the organism, such as the sex chromatin of the inactivated chromosome X
• Identification of sex heterochromatin of chromosomes X and Y is possible in interphase

X Chromatin

• One of the gonosomes X is inactivated in a healthy woman
• In nondividing cells it is in condensed form
• Forms chromatin which is visible in the nucleus as a Barr body
• The number of Barr bodies = number of X chromosomes - 1

Y Chromatin

• Characterized by large heterochromatin blocks on the longer arm of chromosome Y (Yq)
• Can be seen (after staining with fluorochromes) in a fluorescence microscope as a brightly shining point (s.c. F-body)
• The number of F-bodies equals the number of Y chromosomes
• By parallel examination of X and Y chromatin, information about the s.c gonosomal complement of interphase cell can be acquired
• Accurate diagnostics examination of metaphase chromosomes is still essential

Molecular Cytogenetics

• Benefits:
  • Ability to examine any chromosome (or its part) in interphase
  • No in vitro cultivation of cells needed
  • Ability to examine archive material
• Commonly used are:
  • Fluorescent in situ hybridization (FISH)
  • Array comparative genomic hybridization (a CGH), which examines copy number variants

FISH (Fluorescent In Situ Hybridization)

• Based on hybridization of a single-stranded probe with a known sequence of bases (labeled by fluorochrome) with a complementary sequence in genomic DNA
• Examined cells (chromosomes) are fixed on slides
• Extraordinary affinity of molecules is used for labeling probes and fluorochromes, they can recognize each other and bind together even in dilution 1:1,000,000 molecules
• The affinity of biotin and streptavidin is used
• Different types of probes allow for the visualization of:
  • Specific structures of chromosomes like satellites, pericentromeric and centromeric regions, and telomeres
  • Specific sequences in DNA = mutations by "minilocus" probes
  • Whole (single) chromosomes
• Spectral karyotyping (SKY) and multicolor-FISH (mFISH) paint each human chromosome in one of 24 colors
• The combination of diverse labeled probes allows for distinguishing any chromosome

Array Comparative Genomic Hybridization (aCGH)

• High-resolution karyotype analysis solution for the detection of unbalanced structural (deletion and duplication) and numerical chromosomal alterations
• Molecular cytogenetic method for analyzing copy number variations (CNVs)
• Copy number changes at a level of 5–10 kilobases of DNA sequences can be detected
• High-resolution CGH (HR-CGH) arrays are accurate to detect structural variations at a resolution of 200 bp

Why Different Methods

• Different types of chromosomal/DNA changes necessitates different methods
• Different resolution necessities different methods Karyotype:
  • Resolution: > 3 Mb
  • Type of variation detected: Aneuploidy, deletions, duplications, translocations, inversions
  • Example ASD loci: Down syndrome, Turner syndrome, Klinefelter syndrome
  • Benefits: Low costs, widely available
  • Limitations: Low resolutions FISH:
  • Resolution: >1 Mb
  • Type of variation detected: Microdeletion, Microduplications, Translocations, Inversions
  • Example ASD loci: 7q11.23, 15q11-q13, 16p11.2, 17q11.2, 22q11
  • Benefits: Low cost, higher sensitivity to detect small aberrations at specific loci
  • Limitations: Detection of single genes only Microarray (aCGH):
  • Resolution: >15 kb (or less)
  • Type of variation detected: CNV
  • Example ASD loci: SHANK3, NRXN1-4, NLGN3+4, MECP2
  • Benefits: Higher resolution, detection of multiple genes simultaneously, more cost-effective
  • Limitations: Expensive, detection within a specific region, relatively low accuracy, precision, and specificity Sequencing:
  • Resolution: > 1bp
  • Type of variation detected: SNV, Indels, point mutations
  • Example ASD loci: UBE3A, NLGN3, NRXN1
  • Benefits: Highest resolution, entire genome sequenced (gene and exoms)
  • Limitations: Very expensive, identification of abnormalities with unknown clinical significance

Karyotype (Karyogram)

• Summary of chromosome constitution of a person
• Preparation of the complete set of metaphase chromosomes in the cells of a species or in an individual organism, sorted by length, centromere location, and other features
• Individual's collection of chromosomes

Idiogram

• Schematic representation of chromosomes combines data from various banding methods
• Diagram of chromosomes