I. CYTOGENETICS

• Definition:

  • Genetics: The branch of science that deals with the study of heredity and variation.

  • Cytology: The branch of science that deals with the study of structure and function of cell and cell organelles.

  • Cytogenetics: The branch of genetics that deals with the study of the cell and structure and function of chromosomes.

  • Molecular Cytogenetics:

  • Involves the combination of molecular biology and cytogenetic techniques.

  • Utilizes DNA probes labeled with different colored fluorescent tags to visualize specific regions of the genome.

II. KARYOTYPING

• Definition: A laboratory technique used to analyze an individual's chromosomes to detect genetic abnormalities. This involves arranging and photographing chromosomes under a microscope to examine their number, size, shape, and structure.

A. Steps of Karyotyping

1. Sample Collection:

   • Blood (most common)

   • Bone marrow

   • Amniotic fluid (for prenatal diagnosis)

   • Chorionic villus sampling (CVS)

2. Cell Culture & Chromosome Preparation:

   • Cells are stimulated to divide using mitogens.

   • Cell division is halted at metaphase using colchicine.

   • Cells are treated with a hypotonic solution to spread chromosomes.

   • Cells are then fixed and stained (Giemsa stain for G-banding).

3. Microscopic Examination & Analysis:

   • Chromosomes are arranged in pairs (23 pairs in humans, total of 46 chromosomes).

   • Structural abnormalities, missing or extra chromosomes, and large deletions/duplications are analyzed.

B. Types of Karyotyping

1. Conventional Karyotyping:

   • Uses Giemsa staining (G-banding) to detect large chromosomal changes.

   • Diagnoses conditions such as Down syndrome (Trisomy 21), Turner syndrome (45,X), and Klinefelter syndrome (47,XXY).

2. Spectral Karyotyping (SKY):

   • Uses fluorescent probes to uniquely color each chromosome, useful for detecting complex chromosomal rearrangements.

3. Comparative Genomic Hybridization (CGH):

   • Detects copy number variations (CNVs) and small deletions/duplications, more sensitive than conventional karyotyping.

C. Clinical Application of Karyotyping

1. Prenatal Diagnosis:

   • Detects chromosomal disorders such as Down syndrome, Edwards syndrome, and Patau syndrome.

2. Cancer Diagnosis:

   • Identifies chromosomal translocations (e.g., Philadelphia chromosome in CML).

3. Infertility & Recurrent Miscarriages:

   • Detects balanced translocations or aneuploidies.

4. Genetic Syndromes:

   • Diagnoses conditions like Turner syndrome and Klinefelter syndrome.

III. BANDING

• Principle of Banding Techniques:

  • Fall into two groups:

  1. Bands distributed along the length of the chromosome (e.g., G-, Q-band).

  2. Stain specific numbers of bands (e.g., centromeric bands, C-bands, NORs).

• The pattern of bands is numbered from the centromere outward (1 closest).

A. Types of Banding

1. G-Banding (Giemsa Banding):

   • Most common method, used for routine karyotyping.

   • Dark bands = AT-rich, gene-poor regions.

   • Light bands = GC-rich, gene-rich regions.

   • Identifies large deletions, duplications, and translocations (e.g., Down syndrome).

2. Q-Banding (Quinacrine Banded):

   • Uses quinacrine, viewed under UV light, producing a banding pattern.

   • Detects heterochromatin variations.

3. R-Banding (Reverse Banded):

   • Chromosomes heat-treated before Giemsa staining, opposites of G-banding.

   • Best for analyzing telomeric regions.

4. C-Banding (Centromeric Banding):

   • Detects and stains specific regions around centromeres.

   • Useful for identifying Robertsonian translocations.

5. T-Banding (Telomeric Banding):

   • Stains the telomeric region to identify subtelomeric rearrangements.

B. Applications of Karyotyping

• Diagnosing Chromosomal Disorders such as:

  • Trisomies (Down syndrome, Edwards syndrome, Patau syndrome).

  • Sex Chromosome Abnormalities (Turner syndrome, Klinefelter syndrome).

  • Structural Rearrangements (deletions, translocations).

IV. ANEUPLOIDY

• Definition: A genetic condition where a cell has an abnormal number of chromosomes.

  • Normal diploid number is 46 chromosomes; aneuploidy results in missing (monosomy) or extra (trisomy) chromosomes.

A. Types of Aneuploidy

1. Monosomy: Missing one chromosome (e.g., Turner syndrome).

2. Trisomy: An extra chromosome (e.g., Down syndrome).

3. Tetrasomy & Pentasomy: Extra copies of a chromosome (e.g., 48,XXXX).

4. Nullisomy: Missing both copies of a chromosome (usually lethal).

B. Causes of Aneuploidy

• Most commonly caused by meiotic nondisjunction during meiosis I or II, leading to abnormal separation of chromosomes.

  • Age-related, particularly in older mothers.

C. Examples in Humans

• Aneuploidy Conditions:

  • Autosomal Aneuploidy:

  • Trisomy 21 (Down syndrome)

  • Trisomy 18 (Edwards syndrome)

  • Trisomy 13 (Patau syndrome)

  • Sex Chromosome Aneuploidy:

  • Turner syndrome (45,X)

  • Klinefelter syndrome (47,XXY)

  • Triple X syndrome (47,XXX)

  • Jacob's syndrome (47,XYY)

D. Detection of Aneuploidy

• Uses karyotyping, FISH, CGH, and non-invasive prenatal testing (NIPT).

E. Clinical Implications of Aneuploidy

• Miscarriages, developmental disorders affecting intellectual and physical development, and associations with cancer in tumor cells.

V. CHROMOSOMAL ABNORMALITIES

• Occur when there are changes in chromosome number or structure, leading to genetic disorders and developmental issues.

A. Types of Chromosomal Abnormalities

1. Numerical Abnormalities:

   • Trisomy: Breach of the norm, e.g., Down syndrome (Trisomy 21), Edwards syndrome (Trisomy 18), Patau syndrome (Trisomy 13).

   • Monosomy: Turner syndrome (45,X).

2. Structural Abnormalities:

   • Deletions: e.g., Cri-du-chat syndrome (5p deletion).

   • Duplications: e.g. Charcot-Marie-Tooth disease (17p duplication).

   • Translocations: e.g., Chronic Myeloid Leukemia (Philadelphia chromosome t(9;22)).

B. Trisomy Specifics

• Trisomy 21 (Down syndrome):

  • Caused by nondisjunction during meiosis, leading to an extra chromosome 21. Symptoms include intellectual disability, heart defects.

• Trisomy 18 (Edwards syndrome): Symptoms include severe developmental delays, growth retardation.

• Trisomy 13 (Patau syndrome): Severe intellectual disability, organ defects.

VI. MONOSOMY

• Monosomy is a type of aneuploidy where only one copy of a chromosome is present instead of the normal two, resulting in typically lethal conditions.

A. Types of Monosomy

1. Complete Monosomy: Example: Turner Syndrome (45,X).

2. Partial Monosomy: Example: Cri-du-chat Syndrome (deletion of chromosome 5p).

B. Causes of Monosomy

• Mostly due to nondisjunction during meiosis or chromosomal deletions.

C. Detection of Monosomy

• Utilizes karyotyping, FISH, and CGH techniques.

VII. TURNER SYNDROME

• Turner Syndrome (TS) occurs when one X chromosome is missing or structurally abnormal in females, resulting in only 45 chromosomes (45,X).

• Causes: Nondisjunction during meiosis, chromosomal deletions, or mosaicism.

• Clinical Features:

  • Short stature, webbed neck, broad chest, congenital heart defects, ovarian dysgenesis, and normal intelligence but difficulties with spatial reasoning.

• Diagnosis: Karyotyping is the gold standard; FISH detects mosaicism.

• Treatment: Growth hormone therapy, estrogen replacement therapy, and fertility treatment.

VIII. CRI-DU-CHAT SYNDROME

• A genetic disorder caused by a deletion in the short arm of chromosome 5 (5p deletion).

• Clinical Features: High-pitched cat-like cry, low birth weight, facial dysmorphism, and severe cognitive impairment.

• Diagnosis: Karyotyping detects deletions; FISH identifies smaller deletions.

• Treatment: Early intervention programs, medical care for associated issues, and supportive education.

IX. STRUCTURAL OR DELETIONS IN CHROMOSOMES

A. 5q Deletion in Myelodysplastic Syndrome (MDS)

• Characterized by macrocytic anemia and dysplastic megakaryocytes.

• Genetics: Involves deletion of specific genes at chromosome 5q (e.g., RPS14).

B. 17P Duplication in Charcot-Marie-Tooth Disease

• Inherited neuromuscular disorder caused by PMP22 gene duplication.

• Symptoms include muscle weakness, sensory loss.

C. Chronic Myeloid Leukemia (CML) and the Philadelphia Chromosome (t(9;22))

• A type of blood cancer due to a translocation between chromosomes 9 and 22 creating the BCR-ABL fusion gene.

• Phases of CML: Chronic, accelerated, and blast crises.

X. FLUORESCENCE IN SITU HYBRIDIZATION (FISH)

• Definition: A powerful technique used for detecting and localizing specific DNA sequences on chromosomes.

• Applications: Detecting chromosomal abnormalities, microdeletions, cancer diagnosis, prenatal testing.

A. Types of FISH Probes

1. Centromeric Probes: Identify aneuploidies.

2. Locus-Specific Probes: Identify gene deletions/duplications.

3. Whole-Chromosome Paint Probes: Detect translocations.

B. Advantages of FISH

• High sensitivity and specificity, faster results, can be applied to dividing and non-dividing cells.

C. Limitations of FISH

• Cannot detect unknown mutations, more expensive than karyotyping, requires specialized expertise.

D. FISH in CML Diagnosis

• Identifies the BCR-ABL gene fusion necessary for diagnosis and treatment decisions.

XI. COMPARATIVE GENOMIC HYBRIDIZATION (CGH)

• A molecular cytogenetic technique for detecting gains and losses of DNA copy numbers across the genome.

• Applications: Cancer research, prenatal diagnostics, identifying chromosomal imbalances.

A. Types of CGH

1. Conventional CGH: Uses metaphase chromosomes.

2. Array CGH: Utilizes microarrays for higher resolution.

B. Advantages and Limitations of CGH

• Advantages: Genome-wide analysis in a single experiment; detects small deletions better than karyotyping.

• Limitations: Cannot detect balanced rearrangements; array CGH requires specialized equipment.

XII. CHROMOSOME AND GENOME ANALYSIS USING MICROARRAYS

• Applications:

  • Cancer cytogenetics: Identifying genetic abnormalities in tumors.

  • Prenatal cytogenetics: Detects chromosomal abnormalities prenatally.

  • Molecular cytogenetics: Next-generation sequencing and epigenetics applied to cytogenetic studies.

XIII. NEXT-GENERATION SEQUENCING (NGS)

• Definition: A high-throughput technology enabling rapid sequencing of entire genomes or specific regions, revolutionizing genomics and personalized medicine.

• Applications: Diagnosing chromosomal abnormalities, identifying mutations and gene expression patterns, genome sequencing.

A. Key Features of NGS

• Massively parallel sequencing, high sensitivity and accuracy, scalable for different applications, and cost-effective compared to Sanger sequencing.

B. Limitations of NGS

• Expensive setup costs, requires advanced bioinformatics for data analysis, potential errors in specific regions.

XIV. BURKITT LYMPHOMA (BL)

• An aggressive B-cell non-Hodgkin lymphoma characterized by translocation t(8;14) leading to MYC oncogene overexpression.

• Clinical Features: Rapid tumor growth affecting jaws, abdomen, or CNS.

• Diagnosis: Detecting translocation using cytogenetic methods.

• Treatment: High-dose chemotherapy with good prognosis if treated early.

XV. EWING SARCOMA

• Characterized by EWSR1-FLI1 fusion gene resulting from t(11;22).

• Clinical features include persistent bone pain and systemic symptoms like fever and weight loss.

• Diagnosis and Treatment: Confirmed through genetic testing; treated with chemotherapy and surgery for local control.

XVI. GLIOBLASTOMA CYTOGENETICS

• A highly malignant primary brain tumor aggressively associated with chromosomal abnormalities.

• Clinical Implications: Genetic alterations inform treatment and prognosis; targeted therapies under investigation.

XVII. PRENATAL CYTOGENETICS

• Definition: The study of fetal chromosomes to detect genetic abnormalities pre-birth.

• Indications for Testing: Advanced maternal age, abnormal ultrasound findings, recurrent pregnancy losses, family history of chromosomal disorders.

A. Types of Prenatal Cytogenetic Testing

1. Non-invasive: NIPT, Ultrasound.

2. Invasive: Amniocentesis, CVS performing cytogenetic diagnosis.

B. Techniques Used in Prenatal Diagnosis

• Karyotyping for large abnormalities, FISH for rapid detection, CMA for microdeletions.

C. Advantages & Limitations of Prenatal Screening

• High accuracy of NIPT, detection capabilities, limitations regarding small deletions.

D. Ethical Considerations in Prenatal Cytogenetics

• Requires informed consent; implications of false positives or uncertain findings need to be addressed.

XVIII. EPIGENETIC IN FUNCTIONAL CYTOGENETICS

• Refers to heritable changes in gene expression not involving DNA sequence changes, regulated by methylation and histone modifications.

A. Key Epigenetic Modifications

• DNA methylation, histone modifications, and non-coding RNAs function as gene regulators.

B. Techniques Used in Epigenetic Studies

• Includes bisulfite sequencing, ChIP-Seq, methylation-specific PCR.

XIX. MITOCHONDRIAL CYTOGENETICS

• Focuses on the structure and genetics of mitochondrial DNA (mtDNA).

• Common Mitochondrial Disorders: LHON, MELAS, MERRF, and Kearns-Sayre syndrome, often affecting high-energy-demand organs.

A. Detection of Mitochondrial Abnormalities

• Methods like NGS, qPCR, and muscle biopsies for diagnosing mtDNA mutations.

• Treatment Strategies: Mitochondrial Replacement Therapy, dietary adjustments, and symptomatic management.

XX. MITOCHONDRIAL GENETICS & DISORDERS

• Mitochondrial mutations cause various disorders impacting energy metabolism, often requiring sympathetic management strategies.

Reference:

• Viesta, A. (March 2025). Cytogenetics: Nuclear and Mitochondrial Chromosomes [PPT]. BIOCHEM LC 9: CYTOGENETICS Dr. Viesta, A. 03/12/2025 BATCH TANNAWAG 1E