Division of Human Genetics Diagnostic Laboratories

It is important to have reliable clinical information and an accurate family history in order to interpret data from whole exome sequencing (WES) correctly. WES testing is most likely to provide a genetic diagnosis when several family members are analyzed at one time. These items must be included in order to begin the WES process:

• Proband’s sample 
• Maternal sample (for trio testing)
• Paternal sample (for trio testing)
• Additional family members’ samples (following discussion with laboratory)
• Test requisition (all billing and clinical information must be completed)
• Signed informed consent form 
• Family history and pedigree (form is part of test requisition) 
• Detailed patient clinical history / clinical summary or notes
• Summary of previous genetic test results and reports, if available
• Letter of medical necessity describing how medical management will be impacted by the results of this test

Variants that will be discussed in detail in the report:

• Variants that are known to be pathogenic or for which the laboratory has sufficient evidence suggesting pathogenicity in a gene that is suspected to cause the patient’s signs / symptoms

Variants that will be listed in the report:

• Predicted mutation(s) in a gene that is potentially related to the phenotype but for which a specific clinical phenotype has not been previously well defined
• Variants of uncertain clinical significance in genes related to the patient’s phenotype
• Variants that are pathogenic or for which the laboratory has sufficient evidence suggesting pathogenicity in a gene that is medically significant but unrelated to the patient’s presenting symptoms, unless the patient or parent / guardian declines this information

• Variants in genes not believed to be medically significant 
• Variants currently believed to be unassociated with any disease and that are seen in healthy individuals
• Variants that predict an increased risk of diseases, but do not cause a disease by themselves
• Variants identified in research studies and whose relationship with disease is unclear 

Note: Family members who submit samples for comparative analysis will not receive a separate written report.

This laboratory will seek and report pathogenic variants in genes deemed to be of medical value, including those recommended by the American College of Medical Genetics and Genomics (Green et al. 2013).

Secondary findings will not be sought or reported if the patient or patient’s representative chooses not to receive them. For families who choose to receive secondary findings, the patient's report will include this information for all family members who submit blood samples. Family members will not receive a separate report.

Procedure 

ExomeSeq is performed on genomic DNA using the NimbleGen V3 targeted sequence capture method to enrich the whole exome. The whole exome is sequenced on the Illumina HiSeq 2500 sequencing system with 100 base pair (bp) paired-end reads at a minimum coverage of 10X of 95 percent of the target regions. The proband’s exome DNA sequences are mapped and compared to human genome build UCSC hg19 reference sequence.   

Data Analysis 

The laboratory implements dual bioinformatics analyses pipelines that include the use of NextGene V2.3.1 and GATK / Golden Helix V7.7.4 software packages to compare the proband’s sequence to the reference sequence. Assessment of coverage and quality for targeted coding exons of the known protein-coding RefSeq genes is performed. Exome analyses interrogate thousands of genetic variants in a proband and a subset of these is identified as potentially clinically relevant. Sanger sequencing is performed on all variants included in the report.

• This whole exome sequencing test attempts to examine the important coding regions of approximately 20,000 genes in the genome, known as the exome. However, the technical ability to capture and sequence the exome is limited, and currently 85 percent to 92 percent of the entire exome can be evaluated.
• Pathogenic variants may be present in a portion of the genes not covered by this test and therefore would not be identified. Thus, the absence of reportable findings for any gene does not mean there are no pathogenic variants in that gene.
• Certain types of mutations are not detected. Only single base pair changes or small insertions or deletions of DNA are detected. Large deletions, duplications, or rearrangements, mitochondrial genome mutations, trinucleotide repeat expansions, genes with pseudogenes, mutations involved in tri-allelic inheritance, and many epigenetic defects, may not be detected by this test.
• The clinical utility of whole exome sequencing depends on the accuracy of the clinical information provided by the referring physician and the predicted inheritance pattern. DNA sequencing from family members often improves the interpretation of test results.
• Our understanding of the human exome is incomplete at this time.
• Genetic changes identified may not predict severity or age of onset of a particular condition.

• It is highly recommended that patients have genetic counseling before the test is ordered, as they will have an important choice to make regarding which results they wish to know. Understanding the risks and benefits of this testing is important for the patient and his or her family. Genetic counseling after the test is likewise important to aid in the understanding of test results and their implications for the patient and his or her family members.  
• It is the ordering physician’s responsibility to interpret the results from this test within a clinical context.

At least 3 mls whole blood in a lavender top (EDTA) tube. Label the tube with patient’s name, birth date and date of collection. Alternately, 15 mcg of DNA may be submitted.

The time to complete the case preparation and obtain payor precertification is variable. Once payment precertification is obtained, the turnaround time for sequencing and analysis of the exome is three to six months.