Molecular cytogenetics is a field that studies the structure and function of chromosomes at a molecular level. It combines traditional cytogenetics, which involves viewing whole chromosomes under a microscope, with molecular genetics, which analyzes their underlying DNA and RNA. This approach acts as a bridge, allowing for a detailed examination of chromosomal abnormalities that are not visible with standard methods. By using molecular techniques, scientists can pinpoint subtle alterations, helping to diagnose and understand the genetic basis of various diseases and developmental conditions.
The Evolution from Classical Cytogenetics
Classical cytogenetics relied on a technique called karyotyping. In this process, cells were cultured and halted during division when chromosomes are most condensed and visible. These chromosomes were then stained with specific dyes, creating a distinct banding pattern of light and dark areas along each chromosome’s length. Scientists would then photograph these structures and arrange them in a standardized format, or karyogram, based on size and banding pattern.
This method was foundational, allowing for the identification of major chromosomal abnormalities like an extra chromosome, which causes Down syndrome. It provided a complete overview of an individual’s chromosome set, revealing large-scale numerical and structural issues.
The main drawback of karyotyping was its limited resolution, as it could only detect substantial changes involving millions of base pairs of DNA. Smaller genetic changes, such as microdeletions or microduplications, remained invisible, leaving many conditions without a diagnosis. Molecular cytogenetics emerged to address these shortcomings.
Core Techniques Explained
A widely used technique is Fluorescence In Situ Hybridization (FISH). This method uses small, single-stranded DNA fragments called probes, which are designed to be complementary to a specific DNA sequence on a chromosome. These probes are chemically tagged with fluorescent molecules that glow in different colors under a special microscope.
When applied to a patient’s cells, the probes travel to their target location and bind to the matching DNA sequence in a process called hybridization. By observing which parts of the chromosomes light up and how many signals are present, technicians can identify abnormalities like an incorrect number of chromosomes or a translocation.
Another method is microarray Comparative Genomic Hybridization (aCGH), which allows for a genome-wide scan to find regions where DNA has been gained or lost. This technique works by comparing a patient’s DNA sample to a “normal” reference DNA sample. The patient’s DNA is labeled with a green fluorescent dye, while the reference DNA is labeled with a red one.
Both sets of labeled DNA are applied to a microarray slide containing thousands of spots with known DNA segments, where they compete to bind. If the patient has the normal amount of DNA in a particular region, both red and green dyes will bind equally, resulting in a yellow color. If the patient has a deletion (less DNA), the spot will appear more red, and if there is a duplication (more DNA), the spot will appear more green, indicating an imbalance that can be linked to a genetic disorder.
Clinical Diagnostic Uses
In prenatal and postnatal care, molecular cytogenetics provides a detailed look into the genetic health of a fetus or newborn. The techniques are used to screen for conditions arising from an incorrect number of chromosomes, such as Edwards syndrome (Trisomy 18) and Patau syndrome (Trisomy 13). When an ultrasound or blood screen indicates high risk, these tests can provide a definitive diagnosis by analyzing the chromosomal makeup of fetal cells.
The field is also used in cancer genetics, as many cancers are driven by specific chromosomal abnormalities in tumor cells. For example, the detection of the Philadelphia chromosome, a translocation between chromosomes 9 and 22, is a hallmark of Chronic Myeloid Leukemia (CML). Identifying such rearrangements helps diagnose the cancer, determine prognosis, and guide treatment, as some therapies target cells with a particular genetic anomaly.
Molecular cytogenetics also helps identify the genetic causes of developmental delays, intellectual disabilities, and congenital anomalies. Higher-resolution tools can pinpoint the exact genetic imbalance responsible for a patient’s condition, providing families with a diagnosis and a better understanding of the prognosis.
Types of Detected Genetic Abnormalities
Abnormalities identified through molecular cytogenetics are either numerical or structural. Numerical abnormalities involve a change in the total number of chromosomes. The most common type is aneuploidy, which is the presence of an abnormal number of chromosomes, including trisomy (an extra chromosome) and monosomy (a missing chromosome), such as in Turner syndrome.
Structural abnormalities occur when there is a change in the structure or arrangement of a chromosome, which can disrupt the normal dosage of genes. The severity often depends on the size and location of the segment involved. Common types include:
- Deletions, where a segment of a chromosome is lost or deleted.
- Duplications, where a portion of a chromosome is repeated, resulting in extra genetic material.
- Inversions, where a segment breaks off, flips upside down, and reattaches, reversing the gene order.
- Translocations, where a segment from one chromosome detaches and moves to a different chromosome.
A balanced translocation, where no genetic material is lost, may not affect the individual’s health but can pose risks for producing offspring with an unbalanced set of chromosomes.
Scope and Limitations
Molecular cytogenetic techniques have limitations despite their increased resolution. Their primary function is to detect changes in the number or structure of chromosomes. These methods are not designed to identify single-gene mutations, which are alterations in individual DNA base pairs that cause conditions like cystic fibrosis or sickle cell anemia, and require DNA sequencing technologies.
Molecular cytogenetics also cannot detect epigenetic modifications, such as DNA methylation. These changes alter gene activity without changing the underlying DNA sequence itself and are invisible to techniques like FISH and aCGH. Balanced chromosomal rearrangements, where no genetic material is lost, may also go undetected by certain methods like aCGH.
The role of molecular cytogenetics is evolving with the advent of Next-Generation Sequencing (NGS). NGS can provide a higher resolution view of the genome, detecting everything from large chromosomal abnormalities down to single base-pair changes simultaneously. Consequently, molecular cytogenetics is now used as part of a larger diagnostic toolkit, alongside other genetic tests to provide a comprehensive picture of genetic health.