What is Whole Exome Sequencing (WES)?
Whole Exome Sequencing (WES) is a next-generation sequencing (NGS) technique widely used around the world and in our country, involving the sequencing of the “protein-coding regions” of the genome. Exome sequencing includes all DNA segments used for protein production in a human. Although the “human exome” represents less than 2% of the entire genome, this test contains ~85% of the variants associated with known diseases in humans.
“Exome sequencing” is performed to detect variants present in exons. Exome sequencing is a technique used to determine the sequence of DNA in an individual's genetic code and to detect genetic disorders.
How do we diagnose a genetic disease? When do we recommend exome sequencing?
Many different methods are used to diagnose genetic diseases.
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First, detailed family information and the patient's personal history are thoroughly collected.
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Analyzing how the disease is distributed in the family tree is one of the important steps in diagnosis.
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Then a systemic examination and evaluation of dysmorphic features are performed.
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All tests conducted in other clinics before coming to us are reviewed.
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After all these evaluations, if a “pre-diagnosis” is considered, genetic tests are planned for differential diagnosis.
At this stage, if the suspected disease is:
If it is a “single-gene disorder,” molecular methods come to the forefront. If the diagnosis has been clearly made through examination and there is only one gene responsible for the disease, testing is planned for that gene. The method is selected based on the type of mutation most frequently seen in the suspected disease. For some diseases, gene sequencing is prioritized. Sometimes, a method called MLPA is required to primarily investigate deletions and duplications. The decision is made based on the information available about the disease.
In the cases below, single-gene testing is not sufficient.
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Sometimes, diagnosis is not easy. Examination findings may be very subtle.
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Findings are not specific to any disease and may be seen in many different conditions.
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Sometimes the disease is clear, but many genes can cause the same disease (there may be cases where examination cannot distinguish which type it is).
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The patient has a clear disease, but also has one or more additional findings unrelated to that disease. This suggests the possibility of more than one disease.
In such situations, it is very difficult to make a diagnosis by studying only one gene. Especially in “multi-gene related diseases,” studying genes individually can be much more costly and time-consuming compared to large-scale tests like “exome sequencing.”
In such cases, the WHOLE EXOME SEQUENCING – WES method, which involves sequencing all the “protein-coding regions” of 20,000 genes, is preferred.
What are the advantages of the WES method?
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If there is more than one disease or an additional disease that may affect the severity of the main condition, this test allows diagnosis of all conditions at once.
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If the word “atypical” appears frequently in the file—i.e., if there are findings that physicians are unsure about, cannot define, or cannot relate to the disease—there is always a reason. That reason may be mutations in different genes called modifiers that affect the clinical picture or a completely different mutation in another gene causing multiple diseases simultaneously. Exome sequencing enables us to diagnose these possibilities completely.
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Especially in our country, where consanguineous marriage is common, it is not rare to see more than one disease in the same patient. Some patients may even have 4–5 diseases. These results are also very important for determining which genetic tests to conduct in the next children of the family. When we perform this test for couples in consanguineous marriages, the shared carrier genes can be identified, and based on these results, screening tests can be planned for the next pregnancy.
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If the patient's findings are “non-specific—not suggesting a particular disease,” it may be difficult to make a pre-diagnosis. With exome sequencing, we have the chance to make a diagnosis by examining all genes.
What does Whole Exome Sequencing (WES) provide?
Whole Exome Sequencing (WES) is the process of identifying all coding DNA regions (exons) in an individual's genetic material. The advantages of WES are:
Diagnosis of Genetic Diseases:
WES is an effective tool in diagnosing genetic diseases. It is especially used to find and understand the causes of rare genetic diseases.
Identification of Genetic Risk Factors:
WES can assess the likelihood of genetically-based diseases by identifying genetic risk factors in individuals. This helps individuals understand their genetic susceptibility to certain diseases. Additionally, in countries with a high rate of consanguineous marriage like ours, it is a very useful method for pre-pregnancy screening. It detects shared variants in couples and allows us to evaluate pathogenic variants located on the X chromosome in the mother. Thus, it significantly reduces the probability of autosomal recessive and X-linked disorders. It also helps identify genetic diseases that are present in carriers with mild symptoms.
Prediction of Drug Response:
WES can be used to predict how individuals might respond to certain drugs based on their genetic profiles. This enables the development of personalized medicine applications.
Cancer Research:
WES can contribute to understanding the formation and progression of cancer by examining genetic changes in cancer cells. This information can be used to determine individuals’ genetic predisposition to cancer and to personalize treatment strategies.
Genetic Research and Discoveries:
WES is used to discover new genes and understand genetic mechanisms in genetic research. It allows for advances in genetic sciences and the development of solutions for genetic-based problems.
How Is Whole Exome Sequencing (WES) Performed? What Is the Process?
DNA Sample Preparation:
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A suitable biological sample (blood, saliva, tissue) is taken from the individual to obtain genetic material.
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DNA is extracted from this sample in the lab.
Exome Enrichment (Capture):
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Exons, which form only a small part of the genome, are enriched using specially designed probe sets or hybridization methods.
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This step enables more intense sequencing of exon regions by separating them from the rest of the genome.
Sequencing:
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Enriched exons are sequenced using high-throughput sequencing technologies.
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Base pairs in the genetic material are identified, and this information is saved in data files.
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Modern sequencing platforms have the capacity to read millions of base pairs simultaneously.
Data Analysis:
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The obtained sequencing data are analyzed using computer software.
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Genetic variants such as point mutations, insertions, deletions are identified.
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These data are compared with a reference genome to detect potentially significant genetic variants.
Interpretation and Reporting:
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The clinical significance of the analyzed genetic data is evaluated.
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Genetic changes that could potentially be associated with a disease are reported.
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Clinical experts interpret this information by evaluating the patient’s genetic profile.
Whole Exome Sequencing is a powerful genomic research technique used to understand genetic diseases, identify genetic risk factors, and support personalized medicine applications.
Evaluation of Exome Sequencing Data
Evaluation of exome sequencing data is done in light of current scientific knowledge according to individuals’ clinical findings and family histories.
Among approximately 20,500 genes in humans, the association of around 8,000 genes with diseases is currently known. Diseases/findings investigated in the patient are evaluated in terms of findings reported in the literature, and the pathogenicity of detected variants is reported using in silico prediction tools (SIFT, PolyPhen-2, MutationTaster, MetaLR, etc.) and ACMG (American College of Medical Genetics) criteria.
As a result of these studies, it becomes possible to determine treatment methods if a diagnosis is made, screen family members for disease/carrier risk, and prevent this disease in future pregnancies.
Under normal conditions, these analyses take about 1–2 months to complete.
ANALYSIS: Although it may seem simple when described in a few steps, this stage requires in-depth work and extensive experience. Some centers use ready-made analysis software. However, the diagnostic success rate of these programs is not as high as manually performed analyses incorporating clinical data. In our center, we perform analysis by combining the software we developed using our years of experience and clinical data with manual methods. Thanks to this, our diagnostic rates are above the world average.
SUMMARY:
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Exome sequencing allows us to detect variants in the protein-coding region of any gene—not just a few selected ones.
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It is successful in identifying new mutations and carrier states for previously undetectable and unknown diseases.
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It has the potential to enable preventive action before the development of a disease or to initiate treatment for an undiagnosed disease.
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It allows efficient identification of risk factors across a broad spectrum, including genetic diseases and cancer susceptibility genes.
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This method is one of the next-generation sequencing techniques used for diagnosing undiagnosed genetic diseases and conditions involving numerous genes.
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Interpreting exome sequencing data requires identifying patient findings accurately, evaluating all information in the patient’s file, and determining target genes—it requires expertise in genomics, informatics, and clinical medicine.
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Additionally, the test can be interpreted for genetic cancer predispositions, assessing the risk of having a high-risk pregnancy/child in consanguineous couples, and detecting diseases that may require clinical follow-up.
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The aim of this test is to examine about 20,000 genes in a single session, at lower cost and in a shorter time.
For a High-Quality Exome Sequencing Analysis:
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The resolution of data produced in the laboratory
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Detailing the data in bioinformatics, quality assessment, and identification of high-risk variants
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A clinician experienced in patient evaluation reviewing the process with a clinical perspective
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Confirmation of obtained data, family screening, and evaluation of the pathogenicity of identified variants with additional analyses (RNA sequencing, Western Blot, additional tests like X-rays or biochemistry from the patient)
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Availability of statistics derived from the center's own data
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Re-evaluation of undiagnosed or doubtful patients' data at appropriate intervals and re-interpretation with current literature
Prenatal Exome
Interpretation of exome sequencing performed for prenatal diagnosis—i.e., to determine whether the fetus of a pregnant woman is affected—requires even more experience. It can be performed for a finding detected on fetal ultrasound or for an undiagnosed disease in the family. Detected pathogenic and likely pathogenic changes are reported. Multiple databases are screened. If additional findings are detected that may affect the course of pregnancy, alter treatment plans, or worsen clinical conditions outside the fetal findings, the family and clinician are informed in detail and reported accordingly. A report understandable to both the family and clinician is prepared. Genetic counseling must be provided to the family for each report.
Trio Exome
Especially in prenatal exome studies where time is critical, as well as in undiagnosed patients, performing “trio exome” (involving mother, father, and child/fetus) increases diagnostic success and shortens report turnaround times.
Previously, the diagnostic flowchart in prenatal follow-ups after detecting an anomaly in fetal ultrasound involved first performing a rapid test with chromosome analysis. If normal, SNP array was used to detect submicroscopic changes (losses and gains). If still normal, exome sequencing was conducted to detect point mutations. This prolonged diagnosis time in a time-sensitive process. Today, with updates in exome sequencing analysis algorithms, losses and gains can be detected without needing array testing. This method’s ability to detect both single-gene disorders and copy number variations shortens diagnosis times, reduces total costs, and allows for easier work with smaller samples.