Overview

Review of the terminology used in medical genetics Genetics Genetics is the study of genes and their functions and behaviors. Basic Terms of Genetics

Numerical chromosomal aberrations: 

• Aneuploidy: incorrect number of individual chromosomes
• Mosaicism: the presence of ≥ 2 populations of cells within an organism, each with different genomes

Structural chromosomal aberrations:

• Deletions: the loss of a specific chromosome section
• Duplications: multiplication of individual chromosome sections
• Inversions: 180° rotation of a chromosome section and subsequent reintegration into the original chromosome
• Translocations:
  • Exchange of fragments between nonhomologous chromosomes
  • May be balanced (equal) or unbalanced (unequal)

Cytogenetic testing

• Analysis of a specimen for chromosomal changes/abnormalities
• Most commonly used for:
  • Prenatal screening and diagnosis of chromosomal disorders
  • Identification of chromosomal abnormalities related to malignancy 
• Includes:
  • Karyotyping
  • Chromosomal microarray analysis (CMA)
  • FISH

Karyotyping

Definition

Karyotyping, also known as chromosomal analysis, is a common method of producing an image of an individual’s chromosomes to detect:

• Large structural changes in chromosomes
• Aneuploidy

Procedure

• Karyotyping can be performed using various types of samples, including:
  • Blood
  • Bone marrow Bone marrow Bone marrow, the primary site of hematopoiesis, is found in the cavities of cancellous bones and the medullary canals of long bones. There are 2 types: red marrow (hematopoietic with abundant blood cells) and yellow marrow (predominantly filled with adipocytes). Composition of Bone Marrow
  • Amniotic fluid
  • Placental tissue
• Method:
  • Cells from a sample are cultured and stimulated to divide.
  • Colchicine is added to cultured cells → halts cell cycle Cell cycle The phases of the cell cycle include interphase (G1, S, and G2) and mitosis (prophase, metaphase, anaphase, and telophase). The cell's progression through these phases is punctuated by checkpoints regulated by cyclins, cyclin-dependent kinases, tumor suppressors, and their antagonists. Cell Cycle in metaphase → chromosomes are maximally contracted (easier to recognize banding patterns)
  • Chromosomes are then:
    • Stained (allows visualization of banding patterns and chromosome identification)
    • Ordered
    • Numbered (23 pairs)
  • Chromosomes are analyzed according to:
    • Morphology
    • Size
    • Arm Arm The arm, or "upper arm" in common usage, is the region of the upper limb that extends from the shoulder to the elbow joint and connects inferiorly to the forearm through the cubital fossa. It is divided into 2 fascial compartments (anterior and posterior). Arm-length ratio
    • Banding patterns

Uses

Karyotyping is useful for identifying and diagnosing:

• Trisomies and monosomies (e.g., Down syndrome Down syndrome Down syndrome, or trisomy 21, is the most common chromosomal aberration and the most frequent genetic cause of developmental delay. Both boys and girls are affected and have characteristic craniofacial and musculoskeletal features, as well as multiple medical anomalies involving the cardiac, gastrointestinal, ocular, and auditory systems. Down Syndrome/trisomy 21)
• Sex chromosome disorders (e.g., Klinefelter syndrome Klinefelter syndrome Klinefelter syndrome is a chromosomal aneuploidy characterized by the presence of 1 or more extra X chromosomes in a male karyotype, most commonly leading to karyotype 47,XXY. Klinefelter syndrome is associated with decreased levels of testosterone and is the most common cause of congenital hypogonadism. Klinefelter Syndrome/47, XXY)
• Structural abnormalities in chromosomes (e.g., Philadelphia chromosome/reciprocal (9;22)(q34;q11) translocation)

Advantages

• The entire genome can be visualized at once.
• Good sensitivity and specificity for specific conditions
• A wide range of sample types can be used.

Disadvantages

• Can only detect major abnormalities and not small changes
• Interpretation is operator dependent.
• Labor-intensive process

Chromosomal Microarray Analysis

Definition

Chromosomal microarray analysis is a high-resolution technique that utilizes fluorescent tags to identify small chromosomal imbalances.

Procedure

• Can cover the whole genome or can have specific targets (e.g., for known diseases)
• A grid of glass or silicon is covered with thousands of specific nucleic acid sequences (probes).
• Sample and control (or comparative) DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure are:
  • Cut into fragments
  • Labeled with different fluorescent tags
  • Added to the slide
• Hybridized via complementary binding with the probes
• A scanner detects relative gene expression based on the color and intensity of fluorescence (↑ expression → ↑ intensity).

Uses

• CMA provides comprehensive genetic testing for anomalies not detected during karyotyping:
  • Copy number variants:
    • Microdeletions (e.g., DiGeorge syndrome DiGeorge syndrome DiGeorge syndrome (DGS) is a condition caused by a microdeletion at location q11.2 of chromosome 22 (thus also called 22q11.2 syndrome). There is a defective development of the third and fourth pharyngeal pouches, leading to thymic and parathyroid hypoplasia (causing T-cell immunodeficiency and hypocalcemia, respectively). DiGeorge Syndrome/microdeletion at 22q11.2)
    • Microduplications
    • Unbalanced translocations
  • Single nucleotide polymorphisms
• Can also detect aneuploidy
• Used for:
  • Prenatal screening
  • Diagnosis of genetic conditions
  • Forensic analysis
  • Studying diseases and therapies

Advantages

• Can profile the expression of thousands of genes simultaneously
• Can detect very small chromosomal abnormalities

Disadvantages

• Balanced translocations, inversions, and insertions cannot be detected.
• Mosaicism can affect detection.
• Expensive

Fluorescence In Situ Hybridization

Definition

A technique in which a fluorescent DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure (or RNA RNA Ribonucleic acid (RNA), like deoxyribonucleic acid (DNA), is a polymer of nucleotides that is essential to cellular protein synthesis. Unlike DNA, RNA is a single-stranded structure containing the sugar moiety ribose (instead of deoxyribose) and the base uracil (instead of thymine). RNA generally carries out the instructions encoded in the DNA but also executes diverse non-coding functions. RNA Types and Structure) probe is utilized to identify and locate specific genes on chromosomes

Procedure

• A single-stranded DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure probe with a fluorescent dye is used to bind to a specific gene site.
• Probes are hybridized with sample chromosomes on a slide.
• Fluorescence microscopy is used to detect signals.

Uses

• FISH can help directly visualize several chromosomal anomalies, including:
  • Aneuploidy
  • Insertions
  • Translocations
  • Microdeletions (e.g., cri du chat syndrome/microdeletions on the short arm of chromosome 5)
  • Microduplications
• To detect genes associated with malignancy (e.g., HER2 in breast cancer Breast cancer Breast cancer is a disease characterized by malignant transformation of the epithelial cells of the breast. Breast cancer is the most common form of cancer and 2nd most common cause of cancer-related death among women. Breast Cancer)

Advantages

• Improved sensitivity and specificity compared with classic karyotyping
• Can be used for both dividing and nondividing cells
• Multiple probes can be used.

Disadvantages

• Cannot detect point mutations or very small abnormalities
• Not all chromosomal regions have a probe available (at this time).
• The correct probe must be chosen for diagnosis.