Cytogenetic Studies

Introduction

Cytogenetic studies are crucial for understanding chromosomal abnormalities, gene mapping, and genetic disorders. These techniques involve the analysis of chromosomes to detect structural and numerical aberrations. Here, we discuss the various techniques available for cytogenetic studies, their methodologies, and clinical applications.

Karyotyping

Principle:

• Karyotyping involves the visualization of chromosomes under a microscope to identify numerical and structural abnormalities.

Procedure:

1. Cell Culture:
   • Peripheral blood lymphocytes, bone marrow, amniotic fluid, or other cell types are cultured in the presence of mitogens (e.g., phytohemagglutinin).
2. Cell Harvesting:
   • Cells are arrested at metaphase using a mitotic inhibitor (e.g., colchicine).
3. Chromosome Staining:
   • Chromosomes are stained using Giemsa (G-banding), Quinacrine (Q-banding), or other banding techniques.
4. Microscopic Analysis:
   • Chromosomes are analyzed for number, size, shape, and banding patterns.

Applications:

• Detection of aneuploidies (e.g., Down syndrome, Turner syndrome).
• Identification of structural rearrangements (e.g., translocations, deletions).
• Prenatal diagnosis and cancer studies.

Fluorescence In Situ Hybridization (FISH)

Principle:

• FISH uses fluorescently labeled DNA probes to target specific chromosome regions or genes.

Procedure:

1. Preparation:
   • Chromosome spreads or interphase nuclei are prepared.
2. Hybridization:
   • Fluorescent probes are denatured and hybridized to complementary DNA sequences on the chromosome.
3. Detection:
   • Fluorescent signals are visualized under a fluorescence microscope.

Applications:

• Detection of microdeletions and duplications (e.g., DiGeorge syndrome).
• Identification of gene rearrangements in cancers (e.g., BCR-ABL fusion in chronic myeloid leukemia).
• Rapid aneuploidy screening in prenatal samples.

Comparative Genomic Hybridization (CGH)

Principle:

• CGH compares the DNA content of test and reference genomes to detect copy number variations (CNVs).

Procedure:

1. DNA Extraction:
   • DNA is extracted from test and reference samples.
2. Labeling:
   • Test DNA is labeled with one fluorescent dye, and reference DNA is labeled with another.
3. Hybridization:
   • Labeled DNA samples are co-hybridized to metaphase chromosomes or microarrays.
4. Analysis:
   • Fluorescent ratios are analyzed to detect gains or losses in the test genome.

Applications:

• Detection of CNVs in genetic disorders and cancers.
• Identification of chromosomal imbalances in prenatal and postnatal samples.

Spectral Karyotyping (SKY) and Multicolor Fluorescence In Situ Hybridization (mFISH)

Principle:

• SKY and mFISH use multiple fluorescent probes to label each chromosome with a unique color.

Procedure:

1. Probe Preparation:
   • Chromosome-specific probes are labeled with different fluorophores.
2. Hybridization:
   • Probes are hybridized to metaphase chromosome spreads.
3. Imaging:
   • Chromosomes are visualized using spectral imaging or fluorescence microscopy.

Applications:

• Identification of complex chromosomal rearrangements.
• Analysis of marker chromosomes in cancer.
• Detection of cryptic translocations.

Array Comparative Genomic Hybridization (aCGH)

Principle:

• aCGH uses microarrays to detect genome-wide CNVs with high resolution.

Procedure:

1. DNA Labeling:
   • Test and reference DNA are labeled with different fluorophores.
2. Hybridization:
   • Labeled DNA is hybridized to a microarray containing oligonucleotide probes.
3. Data Analysis:
   • Fluorescent intensity ratios are analyzed to identify gains or losses.

Applications:

• High-resolution detection of CNVs in genetic and cancer studies.
• Identification of submicroscopic deletions and duplications.
• Prenatal and postnatal genetic testing.

Polymerase Chain Reaction (PCR)-Based Cytogenetic Techniques

Principle:

• PCR amplifies specific DNA sequences to detect chromosomal abnormalities.

Procedure:

1. DNA Extraction:
   • Genomic DNA is extracted from cells or tissues.
2. PCR Amplification:
   • Specific primers are used to amplify target sequences.
3. Analysis:
   • PCR products are analyzed using gel electrophoresis or sequencing.

Applications:

• Detection of small deletions, duplications, and translocations.
• Diagnosis of single-gene disorders (e.g., Duchenne muscular dystrophy).
• Analysis of microsatellite markers in linkage studies.

Next-Generation Sequencing (NGS)-Based Cytogenetic Techniques

Principle:

• NGS involves high-throughput sequencing to detect chromosomal abnormalities at nucleotide resolution.

Procedure:

1. Library Preparation:
   • Genomic DNA is fragmented, and sequencing libraries are prepared.
2. Sequencing:
   • DNA is sequenced using NGS platforms (e.g., Illumina, Oxford Nanopore).
3. Data Analysis:
   • Bioinformatics tools are used to detect CNVs, structural variants, and aneuploidies.

Applications:

• Comprehensive analysis of chromosomal aberrations.
• Identification of balanced translocations and inversions.
• Detection of low-level mosaicism.

Single Nucleotide Polymorphism (SNP) Arrays

Principle:

• SNP arrays detect CNVs and regions of homozygosity using SNP-specific probes.

Procedure:

1. DNA Extraction:
   • Genomic DNA is extracted and fragmented.
2. Hybridization:
   • DNA is hybridized to an array containing SNP probes.
3. Data Analysis:
   • SNP data is analyzed to identify chromosomal gains, losses, and uniparental disomy (UPD).

Applications:

• Detection of CNVs in genetic disorders.
• Identification of consanguinity or UPD.
• Analysis of genomic imbalances in cancer.

Chromosome Microdissection and Microcloning

Principle:

• Microdissection isolates specific chromosomal regions for further analysis.

Procedure:

1. Chromosome Preparation:
   • Metaphase chromosomes are prepared and stained.
2. Microdissection:
   • Target regions are isolated using micromanipulation.
3. Amplification and Analysis:
   • DNA from dissected regions is amplified and analyzed.

Applications:

• Identification of cryptic chromosomal rearrangements.
• Gene mapping and cloning.
• Characterization of marker chromosomes.

Quantitative Fluorescent PCR (QF-PCR)

Principle:

• QF-PCR uses fluorescently labeled primers to detect aneuploidies and CNVs.

Procedure:

1. DNA Amplification:
   • Specific chromosome markers are amplified.
2. Analysis:
   • Fluorescent PCR products are analyzed using capillary electrophoresis.

Applications:

• Rapid prenatal diagnosis of aneuploidies (e.g., trisomy 21, 18, 13).
• Detection of deletions and duplications.
• Analysis of uniparental disomy.

Clinical Relevance of Cytogenetic Techniques

1. Genetic Disorders:
   • Diagnosis of chromosomal syndromes (e.g., Down syndrome, Turner syndrome).
   • Detection of microdeletions and duplications.
2. Cancer Cytogenetics:
   • Identification of chromosomal rearrangements in leukemia and solid tumors.
   • Monitoring of minimal residual disease.
3. Prenatal Diagnosis:
   • Detection of aneuploidies and structural abnormalities in fetal samples.
   • Analysis of genetic risk in high-risk pregnancies.
4. Reproductive Health:
   • Investigation of recurrent pregnancy loss.
   • Identification of chromosomal abnormalities in infertility.

Limitations of Cytogenetic Techniques

1. Resolution:
   • Conventional karyotyping has limited resolution compared to FISH, CGH, or NGS.
2. Complexity and Cost:
   • Advanced techniques like NGS and SNP arrays are expensive and require specialized expertise.
3. Turnaround Time:
   • Some techniques, such as karyotyping, have longer processing times compared to rapid methods like QF-PCR.