Whole Exome Sequencing (WES) Genetic Test

Whole Exome Sequencing (WES) is a test used to determine the sequence of an individual's DNA and to detect genetic disorders.

What is Whole Exome Sequencing (WES)?

Whole Exome Sequencing (WES) is a Next-Generation Sequencing (NGS) technique widely used worldwide and in our country, involving the sequencing of the "protein-coding regions" of the genome. Exome sequencing covers all DNA segments used for protein production in a human. Although the "human exome" represents less than 2% of the entire genome, this test includes ~85% of 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 order of the DNA sequence in an individual's genetic code and to identify genetic disorders.

How do we diagnose a genetic disease? When do we recommend exome sequencing?

Many different methods are used to diagnose genetic diseases.

• First, detailed family history and the patient's personal medical history are thoroughly documented.
• Examining the distribution of the disease within the family tree is one of the crucial steps in diagnosis.
• Next, a systemic examination and evaluation of dysmorphic features are performed.
• All tests previously performed in other clinics are reviewed.

After all these evaluations, when a "preliminary 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 take precedence. If the diagnosis is clearly established by examination and there is only one gene known to cause the disease, a study is planned for that gene. The method is chosen based on which type of mutation is more frequently observed in the suspected disease. In some diseases, gene sequencing is prioritized. Sometimes, a method called MLPA is required to primarily investigate gains and losses. Decisions are made after researching the specific disease data.

In the following cases, single-gene testing is not sufficient:

• Sometimes diagnosis is not easy. Clinical findings may be very subtle.
• Findings are not specific to any particular disease and can be observed in a wide variety of conditions.
• Sometimes the disease is clear, but there are many genes that cause it (situations where the specific type cannot be distinguished by examination).
• The patient has a clear disease, but has one or more additional findings that cannot be associated with it. In other words, there may be more than one disease present.

In these cases, it is very difficult to diagnose by testing a single gene. Especially in "polygenic diseases," testing genes individually can be much more costly and time-consuming compared to large-scale tests like "exome sequencing."

In these situations, the WHOLE EXOME SEQUENCING – WES method, which sequences the "protein-coding regions" of all 20,000 genes, is preferred.

What are the advantages provided by the WES method?

If the patient has multiple diseases or an additional condition that could affect the severity of the disease, it allows for the diagnosis of all of them with a single test.

If the word "atypical" is used frequently in the file—meaning there are findings that physicians are not fully satisfied with, cannot define, or cannot link to the disease—there is certainly a reason for it. This reason could be mutations in different genes called "modifiers" that affect the clinical picture, or the co-occurrence of multiple diseases due to an additional mutation in a completely different gene. Exome sequencing allows for a complete diagnosis of these possibilities.

Especially in our country, where consanguineous marriage is very common, the rate of multiple diseases occurring in one patient is not low. In some patients, even 4-5 diseases can be observed. These results are also crucial for determining which genetic tests to investigate in the family's future children. Especially in couples with consanguineous marriages, when we perform this test, the genes carried by both spouses are identified, and screening tests for future pregnancies are planned based on these results.

If the patient's symptoms are not "specific" to a particular disease, it may sometimes be difficult to make a preliminary diagnosis. With exome sequencing, we have the chance to diagnose by looking at all genes.

What does Whole Exome Sequencing (WES) provide?

Whole Exome Sequencing (WES) is the process of determining all coding DNA regions (exons) of an individual's genetic material. The advantages provided by WES are as follows:

1. Diagnosis of Genetic Diseases:
   WES is an effective tool in the diagnosis of genetic diseases. It is especially used to find and understand the causes of rare genetic diseases.
2. Identification of Genetic Risk Factors:
   By identifying genetic risk factors in individuals, WES can evaluate the probability of the emergence of genetic-based diseases. This helps individuals understand their genetic predispositions. Additionally, in countries with high rates of consanguineous marriage like ours, it is a very useful method for pre-pregnancy screening. It identifies variants carried by both spouses and allows us to evaluate pathogenic variants on the X chromosome in women. Thus, it significantly reduces the likelihood of autosomal recessive and X-linked diseases. It also enables the identification of genetic diseases present in spouses with mild symptoms.
3. 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.
4. Cancer Research:
   By examining genetic changes in cancer cells, WES can contribute to the understanding of cancer formation and progression. This information can be used to determine individuals' genetic predisposition to cancer and to personalize treatment strategies.
5. Genetic Research and Discoveries:
   WES is used in genetic research for the discovery of new genes and understanding genetic mechanisms. This allows for progress in genetic sciences and the development of solutions for genetic-based problems.

How is Whole Exome Sequencing (WES) Performed? How does the process work?

Whole Exome Sequencing (WES) is generally a process involving the following steps:

1. DNA Sample Preparation:
   
   • An appropriate biological sample (blood, saliva, tissue) is taken from the individual to obtain genetic material.
   • DNA is obtained from this sample in a laboratory environment.
2. Exome Enrichment (Capture):
   
   • Exons, which make up only a small part of the entire genome, are enriched using specially designed probe sets or hybridization methods.
   • This step enables more intensive sequencing by separating exon regions in the genome from other regions.
3. Sequencing:
   
   • Enriched exons are sequenced using high-throughput sequencing technologies.
   • Base pairs in the genetic material are determined, and this information is recorded in data files.
   • Modern sequencing platforms have the capacity to read millions of base pairs simultaneously.
4. Data Analysis:
   
   • The obtained sequence data is analyzed via computer programs.
   • Different types of genetic changes such as genetic variants, point mutations, insertions, and deletions are identified.
   • This data is compared with a reference genome to detect potentially significant genetic variants.
5. Interpretation and Reporting:
   
   • The clinical significance of the analyzed genetic data is evaluated.
   • Genetic changes that could potentially be associated with the disease are reported.
   • Clinical specialists 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 conducted in light of current scientific data according to the individuals' clinical findings and family history.
Today, out of approximately 20,500 genes in humans, about 8,000 are known to have associations with diseases. The diseases/findings investigated in the patient are evaluated in terms of the findings of cases reported in the literature, and the pathogenicities of detected variants are reported according to ACMG (American College of Medical Genetics) criteria using in silico evaluation tool analyses (SIFT, PolyPhen-2, MutationTaster, MetaLR...).
As a result of these studies, it enables the determination of treatment methods if available for the patient's diagnosis, the identification of individuals at risk of disease/carrier status through family screening, and the prevention of this disease in the family's future pregnancies.
The results of these analyses normally take approximately 1-2 months.

ANALYSIS: Although described here in 3-4 points and appearing easy, it is a stage that must be performed in great detail where experience is very important. Some centers use certain ready-made analysis programs. The diagnostic rates of these programs cannot be as high as manual studies incorporating clinical information. In our center, we perform analysis by combining our own developed software with manual methods, incorporating the experience and clinical knowledge we have gained over the years. In this way, our diagnostic rates are above world averages.

IN SUMMARY;

• The exome sequencing technique allows us to determine variants in the protein-coding region of any gene, not just a few selected genes.
• Since all genes are evaluated with exome sequencing, it is successful in identifying new mutations and carrier status for previously undetected unknown diseases.
• It has the potential to increase the ability to act preventatively before disease development or to start treatment for a disease that has not yet been diagnosed.
• Risk factors can be efficiently determined across a wide range of applications, including genetic diseases and cancer predisposition genes.
• This method is one of the next-generation sequencing methods that can be used specifically for the detection of genetic diseases that cannot be diagnosed with clinical and laboratory examinations and diseases where many genes are responsible.
• Interpretation of exome sequencing data requires expertise in genomics, informatics, and clinical medicine to determine target genes by very well-identifying patient findings and evaluating all data in the patient's file.
• In addition, situations such as genetic cancer predispositions, evaluation of risky pregnancy/child status of individuals in consanguineous marriages, and detection of diseases that may require clinical follow-up can also be interpreted with the test.
• The purpose of this test is to examine approximately 20,000 genes at once, with less cost and in a shorter time.

For a Quality Exome Sequencing Analysis;

• Resolution of the data produced in the laboratory,
• Detailing of data in bioinformatics, quality assessment, and identification of high-risk variants,
• Review of the process and clinical perspective by a physician experienced in patient examination and evaluation,
• Confirmation of obtained data, family screening, and additional analyses may be required for the evaluation of the detected variant's pathogenicity (RNA sequencing, Western Blot, requesting and evaluating additional tests like X-rays, biochemistry from the patient),
• The center having statistics obtained from its own data,
• The data of patients who cannot be diagnosed or remain in doubt must be reviewed at appropriate intervals and re-interpretations must be made in light of current literature.

Prenatal Exome

Interpretation of exome sequencing performed for prenatal diagnosis, i.e., to detect whether a fetus is ill in a pregnant woman, requires more experience. It can be performed for a finding detected on fetal USG or for an undiagnosed disease in the family. Detected pathogenic and likely pathogenic changes are reported. Multiple data banks are scanned. If additional findings are detected that would affect the course of pregnancy, change the treatment method, or worsen the clinical picture beyond the fetus's findings, the family and clinician are informed in detail and reported. A report is prepared in a way that the family and clinician can understand. Genetic counseling must be provided to the family for every report.

Trio Exome

Although there is a greater need in prenatal exome studies where time is of the essence, performing "trio exome" consisting of mother-father-child/fetus in exomes performed to diagnose an undiagnosed patient increases diagnostic success and shortens reporting times.

Especially when an anomaly was detected in fetal USG during pregnancy follow-up, the diagnosis flow chart first proceeded to chromosome analysis with a rapid test, then if normal, to SNP array to detect submicroscopic changes (Gains and losses), and if this was also normal, to exome sequencing to detect point mutations. This led to prolonged diagnosis times in a process where we are racing against time. Today, with the arrangements made in exome sequencing analysis algorithms, gains and losses can also be detected without the need for an array test. This method's ability to detect both single-gene diseases and gains/losses significantly shortens our diagnosis times, reduces total cost, and provides the convenience of working with fewer samples.