A copy number variation assay is a laboratory technique that identifies and analyzes changes in the number of copies of specific DNA segments within an individual’s genome. These assays help researchers and clinicians understand how DNA dosage variations influence biological traits and health. They provide insights into the genetic basis of conditions by measuring whether a person has more or fewer copies of certain genes or DNA regions than typically expected.
Understanding Copy Number Variations
Copy number variations (CNVs) are differences in the number of copies of a particular gene or DNA segment among individuals. They involve large DNA sections, from thousands to millions of base pairs. CNVs are a major source of genetic diversity, covering 4.8-9.5% of the human genome.
The two primary types of CNVs are deletions (loss of a DNA segment) and duplications (gain of an extra copy). For example, a duplication might mean three or more gene copies, while a deletion could result in one or zero. Many CNVs are benign, but others can disrupt gene function, alter gene expression, or lead to genomic instability, impacting health.
Clinical Importance of Copy Number Variation Assays
Detecting CNVs is important because these genomic changes impact human health. CNVs are linked to a range of medical conditions, including developmental disorders, neurological conditions, and certain types of cancer. For instance, deletions or duplications of specific genomic regions are associated with conditions such as 22q11.2 deletion syndrome, which increases the risk of schizophrenia and autism spectrum disorder.
In cancer, CNVs drive tumor development and progression through amplification of oncogenes or deletion of tumor suppressor genes. For example, HER2 gene amplification is a known driver in some breast cancers, influencing treatment decisions. CNV assays provide insights into these genetic alterations, aiding diagnosis, prognosis, and treatment strategies. They can also reveal genetic predispositions, supporting personalized medicine.
Methods for Detecting Copy Number Variations
Various laboratory techniques are employed to detect copy number variations, each offering distinct advantages in terms of resolution and scope.
Array Comparative Genomic Hybridization (aCGH)
Array Comparative Genomic Hybridization (aCGH) is a molecular cytogenetic technique comparing a patient’s DNA against a reference. Both are labeled with different fluorescent dyes and hybridized to a microarray chip with thousands of DNA probes. Differences in fluorescence intensity indicate regions where the patient’s DNA has more or fewer copies than the reference, revealing deletions or duplications.
Multiplex Ligation-dependent Probe Amplification (MLPA)
Multiplex Ligation-dependent Probe Amplification (MLPA) is another method using specific DNA probes to detect copy number changes in targeted regions, often at the exon level. In MLPA, pairs of oligonucleotide probes bind to adjacent target sequences; only if adjacent can they be ligated into a single probe. Ligated probes are then amplified using universal primers; the product amount is proportional to target sequences, allowing deletion or duplication detection. MLPA is particularly useful for diagnosing conditions caused by exon-level CNVs.
Quantitative Polymerase Chain Reaction (qPCR)
Quantitative Polymerase Chain Reaction (qPCR) is a highly sensitive method measuring DNA amount in a sample to infer copy number. It involves amplifying a target DNA sequence and a reference with a known copy number. Real-time monitoring determines the target gene’s relative quantity, indicating more or fewer copies than expected. While effective for targeted analysis, it typically requires a reference sample with a known, stable copy number for accurate quantification.
Next-Generation Sequencing (NGS)
Next-Generation Sequencing (NGS) is a technology for CNV detection, offering high resolution and comprehensive genomic coverage. NGS methods detect CNVs by analyzing the depth of sequencing reads across the genome, where more or fewer reads in a region compared to a reference indicate duplication or deletion. Other approaches include read-pair, split-read, and assembly-based analyses. NGS can simultaneously detect various types of genomic variants, making it versatile for identifying CNVs across the entire genome or in targeted gene panels.
Current Applications of CNV Assays
Copy number variation assays are utilized across diverse fields, providing insights into genetic health and disease.
Diagnostic Testing
In diagnostic testing, CNV assays are routinely used to identify genetic causes of unexplained developmental delays, intellectual disabilities, and congenital anomalies. They can pinpoint specific deletions or duplications responsible for a child’s condition, leading to a diagnosis.
Prenatal Diagnosis
Prenatal diagnosis also benefits from CNV assays, detecting CNVs in fetuses. This helps identify potential genetic abnormalities early, aiding genetic counseling and informed decision-making for expectant parents. CNV-Seq, an NGS-based method, has shown superior sensitivity in detecting submicroscopic chromosomal abnormalities compared to traditional karyotyping in prenatal settings.
Cancer Research and Diagnostics
In cancer research and diagnostics, CNV assays identify genomic alterations in tumor cells influencing cancer progression or predicting treatment response. These assays help characterize the genetic landscape of tumors, informing targeted therapy selection and aiding patient outcome prediction.
Pharmacogenomics
Pharmacogenomics, the study of gene-drug response, also leverages CNV assays. CNVs can influence how individuals metabolize certain medications, affecting drug efficacy or increasing adverse reaction risk. Understanding these CNVs helps tailor drug dosages or select alternative treatments for personalized medicine.
Population Genetics and Research
CNV assays also contribute to population genetics and research. They help study the prevalence and role of CNVs in human diversity and disease susceptibility within populations. This provides a broader understanding of how CNVs contribute to the genetic makeup of human populations and their potential adaptive or deleterious effects.