Cytogenetics & Eukaryotic Chromosomes

Cytogenetics

• Cytogenetics is the branch of genetics focused on the study of cell structure and chromosome function
• Molecular cytogenetics combines molecular biology and cytogenetics, often involving DNA probes with fluorescent tags to visualize genome regions

Eukaryotic Chromosomes

• Eukaryotes possess multiple, linear chromosomes, with the number varying by species
• Most eukaryotes have two versions of each chromosome, making them diploid (2N)
• Diploid cells come from haploid (N) gametes fusing to form a zygote
• The zygote will then develop into a new individual
• Chromosome pairs in diploid organisms are homologous (one from each parent)
• Chromosomes with different genes, which do not pair, are nonhomologous
• Animals and certain plants have male and female cells that have distinct chromosome sets
• One sex has a matched pair of chromosomes (XX in human females)
• The other sex has an unmatched pair (XY in human males)
• Autosomes are all chromosomes that are not sex chromosomes

Haploid (N)

• This relates to one copy of genetic material which is subdivided into chromosomes

Diploid (2N)

• This relates to two copies of genetic material which is subdivided into chromosomes

Karyotyping

• Karyotyping uses a uniform chromosome classification system accepted internationally for identification
• A laboratory technique analyzes individual chromosomes to pinpoint genetic abnormalities
• It involves chromosomes being arranged and photographed under a microscope to assess quantity, size, shape, and structure

Steps in Karyotyping

• Sample collection involves blood, bone marrow, amniotic fluid, or chorionic villus sampling (CVS)
• Cells are stimulated to divide using mitogens, and division is stopped at metaphase with colchicine for best visibility
• Cells are treated with a hypotonic solution to swell and spread them, then fixed and stained (e.g., Giemsa for G-banding)
• Microscopic examination & analysis involves chromosomes arranged in pairs (23 pairs, 46 total), with structural abnormalities, missing or extra chromosomes, and large deletions/duplications being noted

Types of Karyotyping

• Conventional karyotyping uses Giemsa staining (G-banding) to find large chromosomal changes, diagnosing conditions like Down, Turner, and Klinefelter syndromes
• Spectral karyotyping (SKY) uses fluorescent probes to uniquely color each chromosome, useful for detecting complex chromosomal rearrangements
• Comparative genomic hybridization (CGH) is more sensitive than conventional karyotyping, detecting copy number variations (CNVs) and small deletions/duplications

Clinical Applications of Karyotyping

• Prenatal diagnosis uses the process to detect chromosomal disorders like Down, Edwards, and Patau syndromes
• Cancer diagnosis helps locate chromosomal translocations like the Philadelphia chromosome in CML
• Karyotyping can uncover balanced translocations or aneuploidies in infertility and recurrent miscarriages
• Genetic syndromes can be diagnosed such as Turner (45,X) and Klinefelter (47,XXY) syndromes

Factors to consider

• Karyotyping is frequent for children without definite non-chromosomal diagnoses
• This can assist with stillbirths and neonatal death (10% are a result of chromosomal abnormalities)
• Infertility problems may be highlighted, such as women presenting with amenorrhea + history of infertility or recurrent miscarriage
• Family history of chromosomal abnormalities may be flagged
• Neoplasia (cancers) are associated with chromosomal abnormalities, more so in advanced age pregnancies (>35 yrs)

Q Banding

• Quinacrine mustard was the first chemical agent used to band chromosomes and viewed under a fluorescence microscope
• Dark and light fluorescence is important to note
• Simple and versatile to use and works where G band is not accepted
• Useful in chromosome heteromorphism
• However, tendency to fade during examination and photo-degradation by UV light are disadvantages

C Banding

• Metacentric: Central centromere with equal-length arms
• Submetacentric: Off-center centromeres with unequal-length arms
• Acrocentric: Centromeres near the ends
• Includes satellites connected by stalks that contain genes encoding rRNA and repetitive sequences
• Allows precise descriptions of band locations, DNA sequences, gene locations, and involvements in chromosomal abnormalities
• Example: BRCA1 gene, 17q21 or human cystic fibrosis gene-7q31.2-q31

Applications of Karyotyping

• Used for detection of chromosomal aberrations
• Used for identifying loss and/or addition of chromosome material
• Helps determine the risk of individual defects/diseases
• Used to Detect aneuploidy
• Used for Pre Birth diagnosis of genetic diseases
• Helps determine gender

Banding in Karyotyping

• Banding identifies individual chromosomes and structural abnormalities through a pattern of light and dark bands based on size, shape, etc

Types of Banding in Karyotyping

• G-banding (Giemsa banding) is most common, used to identify large deletions, duplications, and translocations
• Chromosomes are treated with trypsin and Giemsa dye, where dark bands are AT-rich, gene-poor, and light bands are GC-rich, gene-rich regions
• Q-banding (Quinacrine banding) is where chromosomes are stained with quinacrine and viewed under UV light, producing a similar banding pattern to G-banding
• Helpful for detecting heterochromatin variations and Y chromosome abnormalities
• R-banding (Reverse banding) treats chromosomes with heat before staining with Giemsa, reversing G-banding
• Dark bands are GC-rich, gene-rich, while light bands are AT-rich, gene-poor, best for analyzing telomeric regions
• C-banding (Centromere banding) involves chromosomes treated with alkali and Giemsa, staining centromeric regions and constitutive heterochromatin, useful for detecting centromeric abnormalities
• T-banding (Telomeric banding) focuses on telomeres, needing special staining techniques used for analysis, and is identifies subtelomeric rearrangements

Summary of Types of Banding

• G-banding uses giemsa stain and finds AT-rich/gene-poor regions, or GC-rich/gene-rich regions to check for trisomies, deletions, duplications, translocations in Down/Turner syndrome and CML etc
• Q-banding used quinacrine fluorescent dye fluorescing under UV light similar to G-banding and identifies heterochromatin, used in sex chromosome abnormalities
• R-banding uses giemsa with heat-treatment and finds opposite regions/bands to G, useful for studying telomeric or gene-dense areas
• C-banding uses alkali treatment + giemsa stain, and finds centromeres plus heterochromatin, useful for investigating centromeric abnormalities, isochromosomes, plus Robertsonian translocations
• T-banding uses special giemsa or fluorescence stain to specifically find telomeres that have developmental disorders via subtelomeric rearrangements

Applications of Banding in Medical Genetics

• Banding helps diagnose Chromosomal Disorders like trisomies such as Down, Edwards, and Patau syndromes
• Or Sex Chromosome Abnormalities such as Turner (45, X) or Klinefelter syndrome (47, XXY)
• Or Structural Rearrangements which include deletions (Cri-du-chat syndrome, 5p deletion), and translocations (Philadelphia chromosome in CML)
• Banding can also detect cancer relating to changes with chronic myeloid leukemia (CML+Philadelphia chromosome t[9;22]) and Burkitt's Lymphoma 8;14 resulting in a MYC translocation

Other Factors

• R-banding helps to analyze telomeres
• C-banding is best for studying centromeric abnormalities
• T-banding is used for detecting subtelomeric rearrangements in genetic disorder scenarios
• G-banding is also most used technique plus can detect large scale abnormalities