Clinical exome sequencing is a specialized genetic test that focuses on the protein-coding regions of an individual’s DNA to identify genetic variations linked to disease. It is a powerful method for uncovering the underlying genetic causes of various medical conditions, particularly those that are rare or undiagnosed through conventional testing. This analysis helps healthcare providers gain insights into complex symptoms and guide patient care.
Understanding the Exome
The human genome contains all the genetic instructions for building and operating a person. Within this vast genetic blueprint, specific segments of DNA called genes provide instructions for making proteins, which are the fundamental building blocks and workers of our cells. These protein-coding segments are known as exons. The entire collection of all exons in a person’s genome is collectively called the exome.
Though the exome makes up a small fraction of the human genome, it holds importance for understanding genetic diseases. Approximately 85% of known disease-causing genetic changes occur within these protein-coding regions. Focusing diagnostic efforts on the exome allows for an efficient and targeted search for genetic variations that may explain a patient’s symptoms.
Why Clinical Exome Sequencing is Used
Clinical exome sequencing is used when individuals present with complex or unexplained symptoms suggesting an underlying genetic condition, especially when previous, more targeted genetic tests have not yielded a diagnosis. It helps identify genetic variations responsible for a wide range of inherited disorders.
A primary application is diagnosing rare Mendelian disorders, conditions caused by a mutation in a single gene. For instance, it has been used to diagnose conditions like Kabuki syndrome and Miller syndrome. Finding a genetic diagnosis can end a long “diagnostic odyssey” for patients and their families.
Receiving a specific genetic diagnosis through clinical exome sequencing can influence patient management and treatment plans. For example, identifying the genetic cause of a disease has guided the selection of appropriate therapies, as seen in the treatment of an infant with inflammatory bowel disease. A diagnosis can also inform ongoing screening, aid in reproductive planning, and guide expectations for disease progression. This assessment can also be more cost-effective than performing multiple individual gene tests sequentially.
The Clinical Exome Sequencing Process
The process of clinical exome sequencing begins with collecting a biological sample from the patient, most commonly a blood sample. Other tissue types, such as saliva or skin cells, can also be used, though less frequently. From this sample, the DNA is extracted in the laboratory.
Once the DNA is isolated, it undergoes fragmentation into smaller pieces, typically no larger than 500 base pairs. These fragments are then prepared into a “library” by attaching short adapter sequences to their ends. Exome enrichment, also known as target capture, follows. Here, specialized probes, often biotinylated RNA or DNA molecules, are used to selectively bind to the protein-coding exonic regions of the fragmented DNA.
This hybridization process captures the exome, separating it from the non-coding regions of the genome. The captured exonic fragments are then pulled down, often using magnetic beads, while unwanted non-exonic DNA is washed away. The enriched exome library is then amplified to create enough material for sequencing. Finally, these enriched fragments are sequenced using high-throughput, massively parallel sequencing platforms. This technology generates millions of short overlapping reads, typically between 35 and 100 base pairs long, which are then aligned to a human reference genome to identify variations.
Interpreting Results and Genetic Counseling
After sequencing, the vast amount of raw data generated, which can contain around 20,000 DNA variants per patient, undergoes extensive bioinformatics analysis. This involves several computational steps, including quality control of the reads, aligning them to a reference human genome, and identifying any differences or variations between the patient’s sequence and the reference. These identified variations are then annotated with information from various databases and filtered to prioritize those most likely to be relevant to the patient’s symptoms.
Interpreting these results is a complex task requiring expertise from both laboratory personnel and clinical geneticists. Variants are classified based on available evidence as pathogenic (disease-causing), benign (harmless), or variants of uncertain significance (VUS), where the clinical impact is not yet clear. The test may also uncover “secondary findings,” which are genetic variations unrelated to the original reason for testing but may have clinical implications.
Genetic counseling plays a significant role throughout the entire process. Before testing, genetic counselors provide pre-test counseling, explaining the test’s purpose, its capabilities and limitations, and the types of results that might be obtained, including the possibility of VUS or secondary findings. Following the sequencing, genetic counselors are responsible for explaining the complex results to patients and their families in an understandable way. They discuss the implications of the findings for the patient’s health, potential impact on other family members, and guide them through subsequent steps, such as further testing or management strategies.
Clinical Exome Sequencing Versus Other Genetic Tests
Clinical exome sequencing (CES) offers a middle ground between targeted gene panels and whole-genome sequencing (WGS). Targeted gene panels focus on a specific set of genes known to be associated with a particular condition. These panels are often more cost-effective and have faster turnaround times, making them a good first choice when a specific genetic disorder is strongly suspected. However, their limitation lies in their narrow scope; if the causative gene is not on the panel, the test will not find it, potentially leading to missed diagnoses and the need for further testing.
Whole-genome sequencing, in contrast, involves sequencing nearly all of an individual’s DNA, including both protein-coding and non-coding regions. WGS offers the most comprehensive view of the genome and can detect a wider array of variant types, including structural variations that CES might miss. While WGS has the highest diagnostic rate, its cost and the immense amount of data generated make its analysis more complex and time-consuming. WGS is recommended when CES has not yielded a diagnosis or when a condition is suspected to involve non-coding regions.
Clinical exome sequencing provides a balance between these two approaches. It offers broader coverage than targeted panels by examining all approximately 20,000 protein-coding genes, which account for the majority of known disease-causing mutations. This makes it a suitable choice when a genetic cause is suspected but the specific gene or genes are unknown, or when a patient’s symptoms are complex and could involve multiple genes. CES is typically more cost-effective than WGS while still providing extensive diagnostic utility, making it a frequently adopted option for diagnosing rare genetic diseases.