Chromosome Testing

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 Chromosomes In a prokaryotic cell or in the nucleus of a eukaryotic cell, a structure consisting of or containing DNA which carries the genetic information essential to the cell. DNA Types and Structure
• Polyploidy: multiple sets of chromosomes Chromosomes In a prokaryotic cell or in the nucleus of a eukaryotic cell, a structure consisting of or containing DNA which carries the genetic information essential to the cell. DNA Types and Structure
• 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 Chromosome In a prokaryotic cell or in the nucleus of a eukaryotic cell, a structure consisting of or containing DNA which carries the genetic information essential to the cell. Basic Terms of Genetics section
• Duplications: multiplication of individual chromosome Chromosome In a prokaryotic cell or in the nucleus of a eukaryotic cell, a structure consisting of or containing DNA which carries the genetic information essential to the cell. Basic Terms of Genetics sections
• Inversions: 180° rotation Rotation Motion of an object in which either one or more points on a line are fixed. It is also the motion of a particle about a fixed point. X-rays of a chromosome Chromosome In a prokaryotic cell or in the nucleus of a eukaryotic cell, a structure consisting of or containing DNA which carries the genetic information essential to the cell. Basic Terms of Genetics section and subsequent reintegration into the original chromosome Chromosome In a prokaryotic cell or in the nucleus of a eukaryotic cell, a structure consisting of or containing DNA which carries the genetic information essential to the cell. Basic Terms of Genetics
• Translocations:
  • Exchange of fragments between nonhomologous chromosomes Chromosomes In a prokaryotic cell or in the nucleus of a eukaryotic cell, a structure consisting of or containing DNA which carries the genetic information essential to the cell. DNA Types and Structure
  • May be balanced (equal) or unbalanced (unequal)

Cytogenetic testing

• Analysis of a specimen for chromosomal changes/abnormalities
• Most commonly used for:
  • Prenatal screening Screening Preoperative Care and diagnosis of chromosomal disorders
  • Identification Identification Defense Mechanisms of chromosomal abnormalities related to malignancy Malignancy Hemothorax 
• 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 Chromosomes In a prokaryotic cell or in the nucleus of a eukaryotic cell, a structure consisting of or containing DNA which carries the genetic information essential to the cell. DNA Types and Structure to detect:

• Large structural changes in chromosomes Chromosomes In a prokaryotic cell or in the nucleus of a eukaryotic cell, a structure consisting of or containing DNA which carries the genetic information essential to the cell. DNA Types and Structure
• Aneuploidy
• Polyploidy

Procedure

• Karyotyping can be performed using various types of samples, including:
  • Blood
  • Bone marrow Bone marrow The soft tissue filling the cavities of bones. Bone marrow exists in two types, yellow and red. Yellow marrow is found in the large cavities of large bones and consists mostly of fat cells and a few primitive blood cells. Red marrow is a hematopoietic tissue and is the site of production of erythrocytes and granular leukocytes. Bone marrow is made up of a framework of connective tissue containing branching fibers with the frame being filled with marrow cells. Bone Marrow: Composition and Hematopoiesis
  • Amniotic fluid Amniotic fluid A clear, yellowish liquid that envelopes the fetus inside the sac of amnion. In the first trimester, it is likely a transudate of maternal or fetal plasma. In the second trimester, amniotic fluid derives primarily from fetal lung and kidney. Cells or substances in this fluid can be removed for prenatal diagnostic tests (amniocentesis). Placenta, Umbilical Cord, and Amniotic Cavity
  • Placental tissue
• Method:
  • Cells from a sample are cultured and stimulated to divide.
  • Colchicine Colchicine A major alkaloid from colchicum autumnale l. And found also in other colchicum species. Its primary therapeutic use is in the treatment of gout. Gout Drugs 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 Metaphase The phase of cell nucleus division following prometaphase, in which the chromosomes line up across the equatorial plane of the spindle apparatus prior to separation. Cell Cycle → chromosomes Chromosomes In a prokaryotic cell or in the nucleus of a eukaryotic cell, a structure consisting of or containing DNA which carries the genetic information essential to the cell. DNA Types and Structure are maximally contracted (easier to recognize banding patterns)
  • Chromosomes Chromosomes In a prokaryotic cell or in the nucleus of a eukaryotic cell, a structure consisting of or containing DNA which carries the genetic information essential to the cell. DNA Types and Structure are then:
    • Stained (allows visualization of banding patterns and chromosome Chromosome In a prokaryotic cell or in the nucleus of a eukaryotic cell, a structure consisting of or containing DNA which carries the genetic information essential to the cell. Basic Terms of Genetics identification Identification Defense Mechanisms)
    • Ordered
    • Numbered (23 pairs)
  • Chromosomes Chromosomes In a prokaryotic cell or in the nucleus of a eukaryotic cell, a structure consisting of or containing DNA which carries the genetic information essential to the cell. DNA Types and Structure are analyzed according to:
    • Morphology
    • Size
    • 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)/ trisomy 21 Trisomy 21 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 Sex The totality of characteristics of reproductive structure, functions, phenotype, and genotype, differentiating the male from the female organism. Gender Dysphoria chromosome Chromosome In a prokaryotic cell or in the nucleus of a eukaryotic cell, a structure consisting of or containing DNA which carries the genetic information essential to the cell. Basic Terms of Genetics 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 XXY 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)
• Structural abnormalities in chromosomes Chromosomes In a prokaryotic cell or in the nucleus of a eukaryotic cell, a structure consisting of or containing DNA which carries the genetic information essential to the cell. DNA Types and Structure (e.g., Philadelphia chromosome Philadelphia chromosome An aberrant form of human chromosome 22 characterized by translocation of the distal end of chromosome 9 from 9q34, to the long arm of chromosome 22 at 22q11. It is present in the bone marrow cells of 80 to 90 percent of patients with chronic myelocytic leukemia. Acute Lymphoblastic Leukemia/reciprocal (9;22)(q34;q11) translocation)

Advantages

• The entire genome Genome The complete genetic complement contained in the DNA of a set of chromosomes in a human. The length of the human genome is about 3 billion base pairs. Basic Terms of Genetics can be visualized at once.
• Good sensitivity and specificity Sensitivity and Specificity Binary classification measures to assess test results. Sensitivity or recall rate is the proportion of true positives. Specificity is the probability of correctly determining the absence of a condition. Epidemiological Values of Diagnostic Tests 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 Genome The complete genetic complement contained in the DNA of a set of chromosomes in a human. The length of the human genome is about 3 billion base pairs. Basic Terms of Genetics 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 A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA Types and Structure are:
  • Cut into fragments
  • Labeled with different fluorescent tags
  • Added to the slide
• Probes and sample can hybridized via complementary binding with the probes
• A scanner detects the relative DNA DNA A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA Types and Structure copy number based on fluorescence intensity, showing extra or missing DNA DNA A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA Types and Structure segments.

Uses

• CMA provides comprehensive genetic testing Genetic Testing Detection of a mutation; genotype; karyotype; or specific alleles associated with genetic traits, heritable diseases, or predisposition to a disease, or that may lead to the disease in descendants. It includes prenatal genetic testing. Myotonic Dystrophies 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 Screening Preoperative Care
  • Diagnosis of genetic conditions
  • Forensic analysis
  • Studying diseases and therapies

Advantages

• Can detect copy number variations in thousands of genomic regions at once
• Can identify 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 A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA Types and Structure (or RNA RNA A polynucleotide consisting essentially of chains with a repeating backbone of phosphate and ribose units to which nitrogenous bases are attached. RNA is unique among biological macromolecules in that it can encode genetic information, serve as an abundant structural component of cells, and also possesses catalytic activity. RNA Types and Structure) probe Probe A device placed on the patient’s body to visualize a target Ultrasound (Sonography) is utilized to identify and locate specific genes Genes A category of nucleic acid sequences that function as units of heredity and which code for the basic instructions for the development, reproduction, and maintenance of organisms. DNA Types and Structure on chromosomes Chromosomes In a prokaryotic cell or in the nucleus of a eukaryotic cell, a structure consisting of or containing DNA which carries the genetic information essential to the cell. DNA Types and Structure

Procedure

• A single-stranded DNA DNA A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA Types and Structure probe Probe A device placed on the patient’s body to visualize a target Ultrasound (Sonography) with a fluorescent dye is used to bind BIND Hyperbilirubinemia of the Newborn to a specific gene Gene A category of nucleic acid sequences that function as units of heredity and which code for the basic instructions for the development, reproduction, and maintenance of organisms. Basic Terms of Genetics site.
• Probes are hybridized with sample chromosomes Chromosomes In a prokaryotic cell or in the nucleus of a eukaryotic cell, a structure consisting of or containing DNA which carries the genetic information essential to the cell. DNA Types and Structure 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 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: Anatomy of chromosome Chromosome In a prokaryotic cell or in the nucleus of a eukaryotic cell, a structure consisting of or containing DNA which carries the genetic information essential to the cell. Basic Terms of Genetics 5)
  • Microduplications
• To detect genes Genes A category of nucleic acid sequences that function as units of heredity and which code for the basic instructions for the development, reproduction, and maintenance of organisms. DNA Types and Structure associated with malignancy Malignancy Hemothorax (e.g., HER2 HER2 A cell surface protein-tyrosine kinase receptor that is overexpressed in a variety of adenocarcinomas. It has extensive homology to and heterodimerizes with the EGF receptor, the ERBB-3 receptor, and the ERBB-4 receptor. Activation of the erbB-2 receptor occurs through heterodimer formation with a ligand-bound erbB receptor family member. Targeted and Other Nontraditional Antineoplastic Therapy 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 Sensitivity and Specificity Binary classification measures to assess test results. Sensitivity or recall rate is the proportion of true positives. Specificity is the probability of correctly determining the absence of a condition. Epidemiological Values of Diagnostic Tests 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 Probe A device placed on the patient’s body to visualize a target Ultrasound (Sonography) available (at this time).
• The correct probe Probe A device placed on the patient’s body to visualize a target Ultrasound (Sonography) must be chosen for diagnosis.