Clinical Diagnostic Exome FAQ

Question: What is the process of diagnostic exome sequencing (DES)? 

Answer:  
1. Process

• Sequencing:  Diagnostic exome sequencing (DES) at Ambry involves sequencing of the ~20,000 genes. This is different from whole genome sequencing, and targets the ~1-2% of the protein coding regions of the genome which is expected to cause about 85% of known diseases. The goal of DES is to identify the alteration(s) contributing to the patient’s phenotype through exome sequencing.
   
• Analysis and Review: Once sequencing is complete, the data are processed through Ambry’s bioinformatics pipeline (AVA) and subsequently analyzed by the medical team. Each filtered alteration is reviewed to determine whether it is related to the phenotype of interest. Candidate alterations are chosen for co-segregation analysis.
   
• Reporting:  Each case goes through different levels of medical review, and after final review the report is issued. Each report is unique to the proband and involves literature review and analysis. Reporting may take days to weeks to complete depending on the complexity of the findings.
   
• Turn-around-time (TAT) will vary depending on the test ordered. Additionally, TATs will vary between patients depending on the complexity of the phenotype and number of alterations identified.
   
• Ambry’s diagnostic exome sequencing (DES) test options:
  For all scenarios, provide samples from the entire family (all proband 1st-degree relatives & distant relatives with the same phenotype) from the beginning of testing for co-segregation analysis.
   
• First Tier Exome (FTE):
  Analysis of ~4,000 clinically characterized genes
  Full exome sequencing of a proband
  Familial co-segregation analysis for all positive or uncertain findings
   
• Clinical Diagnostic Exome (CDE):
  Analysis of all captured genes to include novel gene findings
  (~20,000 genes in entire NCBI RefSeq)
  Full exome sequencing of a trio
  Familial co-segregation analysis for all positive or uncertain findings
  REQUIRED: Minimum of a trio; no fetal testing with this option
   
• FTE reflex to CDE
  When FTE results are negative, the test is automatic reflexed to Second-Tier for a complete CDE analysis.

Exome Test Requistion Form - Family Members

Question: What can I expect on the report?

Answer:
1. Primary Findings

• Overall result: positive, likely positive, negative, uncertain
• Summary of alteration
• Summary of gene
• Summary of disease
• Breakdown of number of alterations identified
• Co-segregation results (if applicable)

2. Secondary Findings- Varies depending on preference and age of patient. Report issued separately.  

• Only known pathogenic and likely pathogenic alterations are reported on the secondary findings report. Variants of unlikely clinical significance and benign alterations are not reported. 

Question: When would I order a targeted panel vs. DES?

Answer:
1. When deciding whether to start with one of the targeted panels offered at Ambry vs. DES it is important to consider the yield of testing for each. For example, if the family history shares features of one or several different hereditary cancer syndromes, there may be a clear hereditary cancer syndrome running in the family. In this case it may be more beneficial to start with the targeted next-gen panel such as CancerNext in order to obtain complete gene coverage of the included genes, providing a true negative and rule out these main cancer genes.  

Question: What are the testing requirements for DES?

Answer:
The following are required for testing to proceed and can be emailed to CDE@ambrygen.com, uploaded to Ambry’s secure upload page at www.ambrygen.com/secure-upload/, sent to Ambry with the samples, or faxed to 949-900-5501:

1. A completed test requisition form (for each family member, even those being sent for co-segregation analyses)

2. The Exome Patient Consent Form (included with proband requisition form)

3. Samples from family members (all 1st degree relatives of proband and distant relatives with proband’s phenotype)

4. Family & clinical history information:

• Detailed patient clinical history (in the form of detailed clinic notes)
• A copy of the family history (pedigree)
• Copies of previous test results

Question: What is your policy for reporting secondary findings?

Answer: 
1. ACMG secondary findings minimum list offered as default:

In 2013, the ACMG released “Recommendations on Incidental Findings in Clinical Exome and Genome Sequencing.” The group recommends that laboratories performing diagnostic exome sequencing (DES) actively search and report alterations in genes from among a provided “minimum list” of genes. The list includes 57 genes associated with roughly 25 genetic conditions determined by ACMG to be well-recognized and known to have a strong link of causation. The conditions were chosen if preventative measures and treatments exist. Among the condition types are cancer predisposition risk, later-onset cardiac syndromes, connective tissue syndromes (Marfan Syndrome, Loeys-Dietz Syndrome), and one childhood-onset disease (familial hypercholesterolemia). In following these recommendations, an “ACMG Minimum List” secondary findings report will be issued as a default for all exome orders.

2. Optionally, as part of the standard consent form, patients can decline receipt of all secondary findings. 

3. Additionally, Ambry will retain the original secondary findings options which allow for the expanded secondary finding reports. For patients ordering the Clinical Diagnostic Exome test (CDE), patients have the option to select these expanded reports from among four binned categories (recessive disease carrier status, cancer predisposition, adult-onset disease, and early-onset disease) by completing the “Ambry Patient-Driven Secondary Findings Consent Form”. Please refer to the consent form for details. 

Question: Will you accept proband only cases, when samples from the parents or other first degree relatives are not available? (ex. Adopted patients, etc.)

Answer: 
1. If the proband is only available or if there are less than three first degree relatives in total, the First-Tier only test option is available. 

Question: What are the technical limitations of DES?

Answer:
1. Not all the exons in the genome are targeted: There are many regions of the genome that cannot be captured and sequenced, including many exons. This is due to many factors, including highly repetitive regions and areas of the genome that are highly GC-rich. >97% of exons are targeted.

2. ~10% of Exons that are targeted may not be well covered: The minimum on-target base coverage of any given exome is ~ 90% at 10x, leaving approximately 10% of the exome not covered at levels to reliably call heterozygous variants. Each individual may have slightly different coverage yield distributions within the exome. Given the wide application ranges, clinical sensitivities and specificities for any individual exome are not established.

3. Certain mutation types are not detectable: The assay is limited in the detection of certain mutation types such as large rearrangements, copy number variation (CNV) mutations, mutations involved in tri-allelic inheritance, mitochondrial genome mutations, epigenetic effects, trinucleotide repeat expansions, and X-linked recessive mutations in females who manifest disease due to skewed X-inactivation. (For rearrangements and CNV, CGH-SNP array analysis is recommended prior to exome sequencing).

4. Non-Mendelian Inheritance: Exome sequencing is limited in detection of alterations that are confounded by various factors such as penetrance, variable expressivity, multifactorial disease, epigenetic factors, mitochondrial mutations, phenocopies and UPD.

Question: I understand there are limitations to the mutation types that are detected with DES, but If you see them will they ever be reported?

Answer: 
1. Yes, if an alteration outside of the reportable DES reportable range is detected, the alteration will be Sanger confirmed and reported. If the detected alteration is outside of the technical limits of Sanger sequencing, the results will be discussed with the clinician.

Question: Does Ambry offer re-analysis of sequencing data?

Answer:
1. Yes.

2. Re-analysis of data is available at no charge within two years of sample receipt.  

3. Please note due to the frequent updates to the medical literature and advancements in technology and changes in the patient’s clinical history over time, it may be beneficial to redo the testing in the future.  

4. It is always beneficial to check in with the lab every year for any updates.  

Question: If a variant is reassessed and reclassified will the clinician be notified of the reclassification in order to notify their patients accordingly?

Answer:
1. We are as much invested in reclassifying variants as the clinicians.  Having a definitive interpretation will alleviate our interpretation and find a diagnosis for patients.  There is a continuous effort between the laboratory community and research community to continue to strive to better understand the human genome.  These interpretations and methods are constantly evolving.   

Question: Do you analyze novel genes?

Answer:
1. Yes

2. With the CDE test option we will analyze novel genes.  Novel genes are considered alterations in genes that have not been previously associated with disease.  Due to the complexity of analyzing novel genes, the CDE test option has a longer TAT when compared to the First-Tier only. 

3. If an alteration in a novel gene is found and reported, all efforts will go towards learning more about the gene including coordinating research and functional studies with groups studying the gene, when available.  

Question: Do you perform whole exome sequencing on fetal samples?

Answer:
1. Fetal samples are accepted only for cases of fetal demise.  The First-Tier test option is available for fetal samples.  

2. We currently do not provide WES on current pregnancies. However, we do provide single-site mutation testing during pregnancy. 

Question: Will Ambry provide complete raw variant lists for patients?

Answer:
1. Yes

2. Note: not all alterations on the variant list have been Sanger confirmed and should be used for research purposes only

3. Please complete the “Raw Candidate Alterations” consent form 

Question: Is WES covered by insurance?

Answer:
1. Insurance plans vary with the type of coverage provided for whole exome sequencing. There are many plans that cover the cost of testing. Deductibles will still apply, please contact the billing department for questions and pre-authorization requests. 

2. If you would like to submit for preverification of benefits, please fax the following items to 949-900-5501 ATTN: Exome Pre-auth:

• A completed test requisition form
• Copies of the patient’s insurance cards
• Letter of Medical Necessity
• Family & clinical history information: 

Question: Does Ambry’s test include mitochondrial genome sequencing?

Answer:
1. No, the mitochondrial genome is not part of the captured sequencing for DES.  

Question: If a patient is found to carry a mutation(s), can their family members be tested at Ambry?

Answer:
1. Yes, single site analysis for the alterations identified as causative of the condition of interest is available for family members.  

2. First-degree relatives and other relatives with the same phenotype are covered during co-segregation analysis if the samples are received along with the proband’s sample at the time of testing.