APPLICATIONS OF

CYTOGENETICS IN
MODERN PATHOLOGY
Kyle Vinci P. Solano, RMT, MD, DPSP
Introduction to Genetics & Cytogenetics
• Genetics: Study of heredity, focusing on human genetics and medical genetics.
• Medical Genetics: Focuses on human genetic variation with medical significance.
• Subspecialties: Clinical Genetics, Genetic Counseling, Cytogenetics, Molecular
Genetics, Biochemical Genetics.

Key Genetic Terms

• Gene: DNA sequence that codes for RNA or protein.
• Chromosome: DNA + protein structure carrying genetic information (46 in humans).
• Autosome: Non-sex chromosomes (22 pairs).
• Sex Chromosomes: X and Y (determine gender).
• Mutation: Permanent change in DNA sequence, can be beneficial, neutral, or
harmful.
• Constitutional Mutation: Present in all body cells, passed to offspring.
Chromosome Structure
• Chromatids: Two DNA strands held by a centromere.
• Centromere: Divides chromosomes into p (short) and q (long) arms.
• Telomeres: DNA sequences at chromosome ends, maintain chromosome stability.

Chromosome Types:
• Metacentric: Centromere in the middle.
• Submetacentric: Centromere closer to one end.
• Acrocentric: Centromere near one end, with rRNA gene repeats.
Genetic Terms Continued
• Allele: Variant of a gene (dominant, recessive, codominant).
• Genotype: Genetic makeup (e.g., AA, AO).
• Phenotype: Observable traits (e.g., blood type).
• Homozygous: Identical alleles (AA).
• Heterozygous: Different alleles (AO).
• Hemizygous: Single gene copy (e.g., male with one X chromosome).

Cytogenetics Overview
• Cytogenetics: Study of chromosomes and their structure at the cellular level.
• Chromosomes: 46 chromosomes in humans (22 autosomes, 2 sex chromosomes).
• Karyotype: The chromosomal makeup of an individual.
• Karyogram: Ordered visual representation of chromosomes.
Cytogenetic Techniques
• Cell Culture: Culturing cells to obtain metaphase cells for chromosome analysis.
• Preferred Specimens: Peripheral blood (adults/children), bone marrow
(hematologic disorders), amniotic fluid (prenatal diagnosis).

Culture Methods:
• Suspension Culture: Blood, bone marrow.
• Monolayer Culture: Tissue biopsies, amniotic fluid.
• Mitotic Arrest: Using colcemid to halt cell division at metaphase for analysis.
Clinical Applications of Cytogenetics
• Amniocentesis: Amniotic fluid sample (16-18 weeks of gestation).
• Chorionic Villus Sampling (CVS): Placental tissue sample (10-13 weeks of
gestation).
• Cordocentesis: Fetal blood sample (after 20 weeks gestation).
• Cancer Diagnostics: Identifying chromosomal changes associated with cancer.
• Cytogenetic Testing: Standard for detecting chromosomal abnormalities
(numerical and structural).
Staining Techniques for Chromosome Analysis
• Giemsa/Wright Stain: Positively charged dyes bind to negatively charged DNA,
creating a banding pattern (G-banding).
• Mild Trypsinization: Weakens DNA-protein interactions, enhancing banding
pattern.
• Banding Patterns: Each chromosome pair has a unique pattern, represented as an
ideogram (International System for Human Cytogenomic Nomenclature).
• Special Stains:
• Q-banding: Used for rapid Y chromosome identification; replaced by G-banding
due to transient fluorescence.
• C-banding: Highlights centromere heterochromatin; useful for identifying
dicentric chromosomes.
• R-banding: Reverses G-banding, making telomeres more visible; helpful for
detecting deletions at chromosome ends.
Karyotype Analysis
• Purpose: Identify numerical or structural chromosome abnormalities.
• Step 1: Count chromosomes (normal human: 46 chromosomes).
• Analyze 15-20 cells for accuracy.
• Abnormalities confirmed if 3+ cells show similar chromosomal changes.
• High-Resolution Analysis: Used for detecting small deletions (approx. 3 MB) by
examining prometaphase cells (earlier cell cycle stage).
• Karyogram: Visual representation of chromosomes in a cell (chromosomes
arranged by size, with sex chromosomes placed last).
Computer-Assisted Imaging
• CCD Cameras: Capture metaphase images using video cameras and software.
• Pattern Recognition: Software attempts to arrange chromosomes into a
karyogram; corrections made by cytogenetic technologist.
• Time: A trained technologist typically spends 15-20 minutes to complete a
karyogram.
Fluorescence in Situ Hybridization (FISH)
• Combination of Cytogenetics & Molecular Biology: Detects chromosomal anomalies using
fluorescent DNA probes.

Probe Types:
• Chromosome Painting: Fluorescent DNA fragments covering an entire chromosome.
• Repeat Sequence Probes: Detect chromosome gains/losses.
• Unique Sequence Probes: Identify specific gene deletions or rearrangements.
• Subtelomere Probes: Used to detect small, cryptic chromosomal deletions linked to mental
retardation.

FISH Process:
1. Denature DNA on a slide.
2. Hybridize with a labeled DNA probe.
3. Visualize using a fluorescence microscope.
FISH Applications
• Microdeletions: FISH can detect deletions too small for classical cytogenetics.
• Signal Detection: If the probe binds, there’s no deletion (two signals); if absent, a
deletion exists (one signal).
• Control Probes: Used to ensure hybridization accuracy (e.g., control probe for the
same chromosome arm).
• Mosaicism: Additional cells may be examined if suspected.
• Chromosome Painting: Used to trace the origin of unknown extra chromosome
material.
Multicolor Fluorescence in Situ Hybridization (M-FISH)
• Advantages: Simultaneously hybridizes multiple probes to detect complex
chromosomal rearrangements.
• Up to 3 Colors: Standard FISH can view a maximum of three colors.
• Computer-Assisted Imaging: Allows detection of more colors and complex
rearrangements.
• Prenatal Genetic Diagnosis (PGD): Detects common chromosomal abnormalities
in a single hybridization using multiple probes (e.g., trisomy 13, 18, 21, sex
chromosomes).
• Cancer Studies: Detects chromosomal abnormalities using combinations of
fluorochromes for all 24 chromosomes.
Microarray Technology
• Increased Sensitivity: Detects abnormalities as small as 150 Kb, whereas traditional karyotyping
detects only large changes (~5 Mb).

Clinical Use:
• First-tier diagnostic for developmental disabilities, congenital anomalies, autism, and
neurodevelopmental disorders (2010).
• Postnatal CMA recommended for unexplained developmental delay, intellectual disability, etc.
• Detection: Identifies copy number variations (CNVs) such as deletions, duplications, aneuploidies,
and unbalanced translocations.
• Platforms: Uses aCGH or SNP arrays.
• Reportable Size: 400-600 kb for prenatal, 200-400 kb for postnatal tests.

Limitations:
• Doesn't detect balanced chromosomal rearrangements (e.g., translocations).
• May miss mosaicism or low-level mosaicism.
• Ambiguous findings may require parental follow-up.
Chromosomal Abnormalities
Types:
• Numeric: Changes in chromosome number (e.g., trisomy, monosomy).
• Structural: Changes in chromosome structure (e.g., deletions, duplications).

Numeric Abnormalities:
• Euploidy: Normal (2N, diploidy) or abnormal (triploidy, tetraploidy) chromosome
sets.
• Aneuploidy: Extra or missing chromosomes due to nondisjunction errors in
meiosis/mitosis.
• Trisomy: 3 copies of a chromosome (e.g., Trisomy 21, 18, 13).
• Monosomy: Missing one chromosome (e.g., 45,X, viable monosomy).
• Mosaicism: Multiple cell lines with different chromosome counts.
Mechanisms:
Meiosis Errors:
• First division: Leads to trisomy or monosomy.
• Second division: May produce gametes with extra or missing chromosomes.

Rescue Mechanisms:
• Uniparental Disomy (UPD): Both chromosomes from one parent.
• Isodisomy: Two copies of a chromosome from one parent.
• Heterodisomy: Two different copies from one parent (e.g., grandparental inheritance).

Microarray in Genetic Diagnosis

• Advantages: Identifies smaller genetic lesions missed by other tests.
• Limitations: Does not detect balanced rearrangements. NGS can offer further insights
when CMA is inconclusive.
•
Overview of Chromosome Rearrangements
• Chromosomal Structure: Chromosomes undergo recombination during
meiosis/mitosis; errors may lead to structural abnormalities.
Types of Rearrangements:
• Balanced: Chromosomal material is rearranged but not lost/duplicated. Clinically
usually benign but may affect meiotic segregation.
• Unbalanced: Loss/duplication of chromosomal material, leading to clinical issues
such as developmental delay or mental retardation.
Types of Structural Abnormalities
1. Deletions:
• Loss of a chromosome segment, leading to partial monosomy.
• Can be terminal (end of the chromosome) or interstitial (internal segment).
• Larger deletions = more severe clinical impact.

2. Duplications:
• Extra copy of a chromosome segment, causing partial trisomy.
• Can be terminal or interstitial.
• Often reciprocal of a deletion (duplication of lost material).
• Larger duplications = greater genomic imbalance.

3. Inversions:
• Reversal of a chromosome segment, requiring at least two breaks.
• Paracentric inversion: Breaks in the same chromosome arm.
• Pericentric inversion: Breaks on both sides of the centromere.
• May be balanced but cause clinical issues if gene disruption occurs.
4. Translocations:
• Rearrangement between non-homologous chromosomes.
• Often balanced but can cause clinical problems if important genes are disrupted.
• Risks during meiosis due to abnormal segregation patterns (adjacent-1, adjacent-2, or 3:1).
• Robertsonian translocation: Fusion of acrocentric chromosomes (e.g., chromosomes 13 and 14), increasing
trisomy risk (e.g., trisomy 13 or Down syndrome).

5. Isochromosomes:
• Misdivision of the centromere, producing two copies of one chromosome arm and loss of the other.
• Example: Isochromosome of Xq in Turner syndrome (trisomy for X long arm, monosomy for short arm).

6. Ring Chromosomes:
• Form when both telomeres of a chromosome are lost and the chromosome circularizes.
• May be unstable but can be stably transmitted in some cases.

7. Marker Chromosomes:
• Small or ambiguous chromosomes with a centromere that may be stably transmitted.
• Can indicate partial trisomy/tetrasomy, and may be identified by microarray.
Clinical Implications of Structural Abnormalities
• Unbalanced abnormalities: Associated with clinical symptoms such as developmental
delay and mental retardation.
• Balanced abnormalities: May have little or no clinical effect but can cause meiosis
errors in carriers, increasing the risk of chromosomally abnormal offspring.

Chromosome Nomenclature
• Basic Structure: Describes the number of chromosomes, sex chromosome composition,
and any structural abnormalities.
• Example: 46,XX (normal female), 45,X/46,XX (mosaicism).
• Numerical abnormalities: Trisomy (+) or monosomy (−) notation (e.g., 47,XX,+13 for
trisomy 13).
• Structural Abnormalities: Denoted by abbreviations (e.g., del, dup, t, inv).
• Example: 46,XX,del(4)(p15) (deletion of chromosome 4 at band p15).
• Translocation Example: 46,XY,t(4;9)(q21.2;p22) (translocation between chromosomes 4
Prenatal Cytogenetics:
Chromosomal Abnormalities:
• 1 in 13 conceptuses has a chromosomal abnormality; however, most do not result in live births.
• Common chromosomal errors in spontaneous losses include trisomy 16 and 45,X.

Risk Factors:
• Advanced maternal age increases the likelihood of chromosomal abnormalities, especially trisomy
21 (Down syndrome).
• Prenatal testing is frequently recommended for older mothers, family history of abnormalities, or
abnormal prenatal screening results.

Prenatal Testing Techniques:

• Karyotype analysis and FISH are used for identifying common aneuploidies (chromosomes 13, 18,
21, X, and Y).
• Noninvasive prenatal testing (NIPT) since 2012 screens for fetal aneuploidies using cell-free fetal
DNA from the mother’s blood.
• NIPT provides high accuracy for trisomies 13, 18, and 21 but has higher false-positive rates for
Professional Recommendations:
• The American College of Medical Genetics and Genomics (ACMGG) and ACOG
recommend NIPT for aneuploidy screening.
• NIPT is a screening tool, not diagnostic; abnormal results require confirmation by
karyotype, FISH, or microarray.

Postnatal Cytogenetics:
Newborn Testing:
• Approximately 0.6% of newborns have chromosome abnormalities.
• Karyotype and FISH can confirm diagnoses for syndromes, ambiguous genitalia, and
anomalies linked to chromosome abnormalities.

Death in Infancy:
• Cytogenetic analysis can help explain neonatal death and should be correlated with
autopsy findings.
Cancer Genetics & Cytogenetics in Oncology
Role of Cytogenetics in Oncology:
• Cytogenetic analysis, especially of bone marrow and blood, is crucial in
diagnosing leukemias and lymphomas.
• Chromosome abnormalities (e.g., translocations) are directly linked to cancer
progression and response to therapy.
• WHO classification (latest in 2017) integrates genetic findings as core diagnostic
elements.

Karyotype Analysis in Cancer:

• Prognosis correlates with type/number of chromosomal abnormalities.
• Successful treatment is marked by cytogenetic remission, indicated by a return to
a "normal" karyotype.
• Disease progression may show “karyotype evolution,” with increasing
FISH (Fluorescence In Situ Hybridization) in Oncology:
• FISH provides rapid analysis of both metaphase and interphase cells, enhancing
diagnostic accuracy.
• Key applications include detecting BCR/ABL1 fusion in the 9;22 translocation
associated with CML, AML, and ALL.
• Variants of FISH patterns indicate complex chromosomal events, useful for
tracking residual disease.

Additional FISH Applications:

• Detection of specific chromosomal aneuploidies (e.g., trisomy 12 in CLL).
• Monitoring bone marrow transplants using sex chromosome markers.
• Multicolor FISH helps reveal the high degree of rearrangement in cancer cells.
Chromosomal Aneuploidy Syndromes
Autosomal Aneuploidies:
• Trisomy 21 (Down Syndrome): Most common, with features like hypotonia, facial abnormalities,
heart defects; increased cancer risk.
• Trisomy 13 (Patau Syndrome): Rare, marked by cleft palate, polydactyly, usually fatal within the first
month.
• Trisomy 18 (Edwards Syndrome): Similar to Patau syndrome in severity, with features like small jaw,
low birth weight, often fatal early in life.

Sex Chromosome Aneuploidies:

• Generally milder due to X inactivation and limited Y chromosome genes.
• 47,XXX Females and 47,XYY Males: Often undetected, tall stature, sometimes learning difficulties.
• Klinefelter Syndrome (47,XXY): Males with taller stature, hypogonadism, infertility; mosaics
(47,XXY/46,XY) may retain fertility.
• Turner Syndrome (45,X): Females with short stature, gonadal dysgenesis, variable phenotype; some
fertility possible in mosaic cases.
Other Sex Chromosome Anomalies:
• Disorders involving the SRY gene on the Y chromosome can result in ambiguous
genitalia or atypical development.
• Examples include XX males from cryptic X-Y translocations, or androgen
insensitivity leading to a female phenotype with an XY complement.
Microdeletion and Contiguous Gene Syndromes
Microdeletion Definition:
• Small deletions (>500 kb) affecting several genes; detected via FISH or microarray
rather than karyotyping.
• Contiguous gene syndromes arise when deletions span multiple neighboring
genes, leading to complex phenotypes.

Key Syndromes:
Miller-Dieker Syndrome:
• Caused by a deletion on chromosome 17p13.3.
• Symptoms: lissencephaly (smooth brain), craniofacial anomalies.
• Involves multiple genes; smaller deletions only affect the LIS1 gene, causing
isolated lissencephaly.
Prader-Willi Syndrome vs. Angelman Syndrome:
• Both involve deletion on chromosome 15q11.2, differing by genetic imprinting.

• Prader-Willi: hypotonia, obesity, small hands/feet, developmental delays,

behavior issues.

• Angelman: severe intellectual disability, hyperactivity, seizures, ataxia,

inappropriate laughter.
• FISH detects the deletion in 80–85% of cases.

Williams Syndrome:
• Deletion of elastin gene on chromosome 7q11.23.
• Symptoms: cardiac issues, skin aging, hoarse voice, unique behavior.
• Behavioral traits linked to adjacent gene deletions, not just elastin.
WAGR Syndrome:
• Deletion on chromosome 11p13; affects multiple genes.
• Symptoms: Wilms tumor, aniridia, genitourinary defects, intellectual disability.
• Phenotype varies with deletion size; some children with aniridia may develop Wilms tumor.

Velocardiofacial Syndrome (22q11.2 Deletion Syndrome):

• Common microdeletion; diverse symptoms include feeding issues, cardiac defects, facial dysmorphologies.
• Caused by misaligned chromosomal recombination, can lead to deletions or duplications with distinct
phenotypes.

Fragile X Syndrome:
• Second leading cause of mental retardation, involving X chromosome instability.
• Caused by a trinucleotide repeat expansion in the FMR1 gene.
• Detected via molecular analysis rather than cytogenetic testing.

Chromosomal Breakage Syndromes:

• Group of autosomal recessive disorders characterized by chromosome fragility and instability.
• Caused by mutations in DNA repair genes, leading to cancer susceptibility due to accumulation of DNA
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