Diagnostic Exploratory Testing

Presentation

Expiration Date: December 13, 2026

Presenter

Sarah Barnett, M.S., CGC

Division of Laboratory Genetics and Genomics
Mayo Clinic, Rochester, Minnesota

Nicole Boczek, Ph.D.

Assistant Professor of Laboratory Medicine and Pathology
Division of Laboratory Genetics and Genomics
Mayo Clinic, Rochester, Minnesota

Transcript

Introduction

Hi. Thank you so much for joining us today. 

My name is Nicole Boczek, and I am an assistant professor and laboratory director in the Department of Laboratory Medicine and Pathology and serve as a co-director within the Laboratory of Genetics and Genomics here at Mayo Clinic. I specialize in hereditary disorders, more specifically, diagnostic exploratory testing, which includes exome and genome sequencing, chromosomal microarray, and congenital chromosomes. Which brings us to our topic today, which we will be discussing the diagnostic exploratory testing suite available at Mayo Clinic Laboratories.

And I am Sarah Barnett, a genetic counselor with 12 years of experience working closely with Dr. Boczek to support those same specialty areas at Mayo Clinic for the past seven years. Prior to my time in the lab, I was a genetic counselor in a general genetics clinic ordering these same tests that we’re going to be discussing today.

Disclosures

We have nothing to disclose.

Learning objectives

With the launch of clinical genome sequencing in the last year here at Mayo Clinic Laboratories, we wanted to take this opportunity to look back on what diagnostic exploratory testing means in our lab, why it’s so important to the field of many specialty practice areas, and help our colleagues determine which testing may be the most valuable for a given patient.

What is exploratory testing? Which tests are considered exploratory?

The tests that fall into our diagnostic exploratory menu here at Mayo Clinic include congenital chromosomes, or karyotype, chromosomal microarray, whole exome sequencing, often shortened to “exome” or “WES,” and whole genome sequencing, often shorted to “genome” or “WGS.” We call these tests exploratory, not because they are research tests — they are very much clinic diagnostic tests — but because they aren’t targeted at a single variant, single gene, or even a single set of genes, as in panel testing. These tests represent true clinical grade tests, and while single-gene and panel testing are still relevant today we are asking the question more and more, “When do we use diagnostic exploratory testing, especially exome and genome?”

Information about new gene-disease associations, new variants in genes that cause the protein they make not to work correctly, and whole new ways to look at the genome are evolving rapidly. We’ve been doing karyotypes for 50 years looking at aneuploidy, large copy number variation and structural rearrangements. In the last 15 to 20 years with the introduction of chromosomal microarray the size of the copy number variants we can see got smaller. And in the last 10 years, we gained the ability to sequence the coding regions of our genome, the exome. Which really accounts for just 1%-2% of the genome where we can detect single nucleotide variants in addition to small, medium, and large copy number variants. In the last three to five years, whole genome sequencing has emerged as a clinical test in which the entire genome is sequenced, including those intronic regions that we were missing with whole exome, and the resolution of copy number variants that can be detected got smaller on both exome and genome. With genome, we gained the ability to detect repeat expansions, variants in challenging regions like the spinal muscular atrophy locus, and mitochondrial variants with a single test. And diagnostic exploratory testing is still evolving with continued improvements to bioinformatics and variant calling, RNA sequencing, proteomics and whole methylome on the horizon!

Why perform exploratory testing?

Now that we’ve covered what exploratory testing is we want to focus on the why. And here we are mostly thinking about exome and genome. In a clinical practice guideline published in 2021, the American College of Medical Genetics and Genomics recommends exome and genome as first-tier testing for patients with one or more congenital anomalies or a neurodevelopmental disorder. Additionally, in a practice guideline in 2022, the National Society of Genetic Counselors recommends the use of genome sequencing as the genetic testing of choice for individuals with unexplained epilepsy. The reasons behind these recommendations are multifold. Literature shows that for most disease states the diagnostic yield on an exome or genome is higher than panel-based testing, up to 56% overall, because we can provide diagnoses for patients with newly described or very rare disorders that aren’t covered by any panel test. Additionally, exploratory testing allows us to reveal as yet undiscovered gene-disease relationships, or expanded phenotypes for known disease genes. When used as first-tier testing, exome and genome significantly reduce the time the patient and family spend in the “diagnostic odyssey” phase of rare diseases, where years may pass with many different medical visits, procedures, and delays in treatment. In patients who do receive a diagnosis, we see direct changes to patient care in up to 65%, and for those that do not, exome and genome allow for reanalysis of previously generated data over time which also increases the yield. Here we are displaying the percent diagnostic yield achieved on the x-axis for each methodology, broken down by variant type, indicated by the colored sections, with reanalysis being indicated in orange.

Genome, specifically, allows for testing of multiple variant types not typically provided with panels or exome, thus resulting in up to 30% of cases receiving a diagnosis when genome is performed after prior exome testing. Additionally, all diagnostic exploratory testing allows for detection of secondary findings, or variants in genes with clear medical actionability or treatment guidelines even if they aren’t related to the original reason for testing.

Diagnostic exploratory testing at Mayo Clinic

These types of exploratory tests are offered by many different laboratories. However, not all tests are built the same; therefore, we would like to bring you along to better understand what our exploratory tests do here at Mayo Clinic.

Diagnostic exploratory test comparison

As a means to visualize and highlight the complexities of what each exploratory testing methodology can detect, we created this Venn diagram. I’d like to draw your attention to a few things. First, genome detects nearly all variant types except for balanced rearrangements, those currently only being detected by karyotype testing which is primarily used for counseling reproductive risk, and large mitochondrial CNVs, which at Mayo Clinic are best detected using the specific mitochondrial genome sequencing assay. Second, in contrast to microarray, karyotype and mitochondrial DNA testing or other panel testing, exome and genome analysis employ a phenotype-driven approach which we will discuss a bit more later, and those assays both allow for familial samples to be incorporated into the initial analysis to help prioritize and classify variants. The other primary differences highlighted here are size of copy number variants detected by each assay and some specialized variant types. 

Please feel free to pause this presentation and review this diagram further and reach out with questions. I will call your attention to the bottom of the figure, where you can review the CNV resolution by methodology but will note it is hard to compare sizes as the methodology for copy number calling varies between these strategies.

Advantages of exome & genome

When WES or genome is performed, tens of thousands to hundreds of thousands of variants are identified in every patient. Manual review of all variants is not possible or necessary. Thus, laboratories must design variant filtering strategies to reduce the number of variants for manual review of the most clinically impactful variants. At Mayo Clinic, in order to bring a small set of reportable variants to the forefront, we leverage both artificial intelligence software algorithms and a manually defined set of filtration strategies, which we have refined and customized based on Mayo Clinic’s nearly 10 years of clinical exome and genome experience. Our approach is multifaceted, with the four main strategies displayed in this figure. We take a phenotype-driven approach by reviewing variants in genes on a patient specific gene list generated using clinical features provided by medical record review or the ordering clinician. We take a family-based approach when comparators are provided to ensure we review variants that are de novo, or compound heterozygous, or shared between affected family members. We also review variants that are molecularly interesting, for example, being predicted to cause loss of function, or variants that are previously reported as disease-causing, and finally, we review predicted pathogenic or likely pathogenic variants in a set of medically actionable genes as defined by the American College of Medical Genetics and Genomics if the patient does not opt out.

I’d now like to hand it over to Dr. Boczek.

Post-resulting

Thanks Sarah. Now that we have given you a little bit more information about the technical aspects of our diagnostic exploratory testing, we would like to change our focus to tell you how patient-centered we are here at Mayo Clinic. We continue to provide care after the clinical report is received through the many facets you can see here. As part of our testing suite, we perform familial variant testing which can be completed on either similarly affected or unaffected family members, depending on the circumstance and question at hand. Based on the classification of the variant of interest, sometimes this testing can be performed free of charge. We also have patient management options which include referral to Mayo Clinic's Department of Clinical Genomics if there's assistance needed with management of complex conditions.

It is very important to note that Mayo Clinic has a full-service laboratory, meaning we have options for additional studies such as biochemical follow-up. We also have a full FISH and Chromosome Laboratory, so if structural characterization is necessary to disambiguate a result, or to predict familial risk, this can be performed here at Mayo Clinic. This particular service is not offered by many other laboratories, requiring the physician or ordering provider to work with multiple labs and communicate complex information between laboratories. All of this is simplified using Mayo Clinic Laboratories as our full suite of follow-up testing can be completed with a streamlined add-on process. 

Our diagnostic exploratory suite of tests also participates in both internal and external collaboration activities. We submit all of our variants for classifications in ClinVar on a routine basis to help aid in variant classification. We also use GeneMatcher to aid in novel gene-disease discovery to ensure your patients are getting cutting edge diagnoses. We strive to be active in the community to learn more and to better help provide as many diagnoses for as many patients as possible.

As part of our diagnostic exploratory service, specifically for exome and genome, we also offer reanalysis. Reanalysis typically occurs at least one year after report release, or if the patient’s phenotype has drastically changed. The first reanalysis for exome and genome sequencing is performed free of charge. Due to external research opportunities, we also have the capability to return raw data. And finally, something that's really beneficial here at Mayo Clinic is that we have a dedicated staff of genetic counselors as well as lab directors that are available and just a phone call away. These individuals can help answer many questions including supporting ordering questions and appropriateness of testing as well as aid in personal interpretation of results.

What does Mayo Clinic bring?

In addition to what we do post-resulting, we have a huge breadth of diagnostic exploratory expertise here at Mayo Clinic. To highlight, in our diagnostic exploratory practice, our combined set of eight lab directors, four genetic counselors, and one variant scientist have over 167 years of experience and have published over 400 articles. You can see a list of the many noteworthy publications in the diagnostic exploratory space that we have participated in here, including many best practice level guidance documents, and variant classification guidance. So, this is a space we are very comfortable in and have a lot of expertise in, and we can be trusted to interpret results for your patients. We also very actively contribute in many different committees at outside institutions including ClinGen, CAP, and ACMG. We participate in these committees to not only bring knowledge back to the clinic, but also to share our breadth of clinical knowledge with other laboratories and institutions across the United States and internationally.

Application of knowledge and expertise – Two case examples

Now that we've given you a background on the testing we have here at Mayo Clinic, what we offer in the post resulting space, and our team’s specific expertise in the diagnostic exploratory testing space, we would really like to give you two case level examples to showcase our knowledge base.

Case 1: Unidentified intellectual disability

Our first case includes a 12-year-old male with unidentified intellectual disability. This individual also has some additional features as described here. In this particular family, the mother, the father, and the older brother all shared similar features with variable severity. This particular individual did have a previous microarray which revealed a duplication of uncertain significance. The microarray findings were unable to resolve the cause of intellectual disability in this family. In this particular case, the provider decided to order whole exome sequencing on the affected child and similarly affected mother and brother for analysis.

Exome sequencing revealed a pathogenic 21-kilobase deletion involving two genes, including the PSMD12 gene. Loss of function variants in this particular gene are associated with autosomal dominant StankiewiczIsdor syndrome (STISS) which has a lot of phenotypical overlap with this patient and his family. It is important to note the mother and the brother also had the same deletion showing segregation of the variant with this phenotype. Some noteworthy things to discuss about this case is that the PSMD12 deletion was below the resolution of chromosomal microarray, so microarray testing alone would not have led to this diagnosis, however this event is detectable by both exome and genome sequencing.

I also want to highlight what we discussed previously in this presentation that exome and genome sequencing can capture the same events as microarray. And in this example, the duplication of uncertain significance that had been previously reported by microarray was also detected by exome sequencing.

Case 2: Unexplained sudden death in infant

Our second case involves a tragic unexplained sudden death in an infant who presented with the multiple anomalies as highlighted here. In this case, there were no similarly affected family members. For this analysis, the provider decided to move forward with genome sequencing of a traditional trio including the mother, the father, and the child. In this particular case, a de novo frame shifting variant in the MECOM gene was identified by genome sequencing. Pathogenic variants in MECOM have been associated with dominant radioulnar synostosis with amegakaryocytic thrombocytopenia, which has a wide range of phenotypes, all of which did not completely overlap with our patient. However, recent studies have shown that loss of function variants can lead to more severe presentations, including infant loss, which aligns with this particular circumstance. It is important to note, bone marrow failure conditions were not necessarily on the original differential for this case. In these types of circumstances, exome and genome sequencing provide many advantages, as we were able to come to a diagnosis that may not have otherwise been considered due to a novel presentation of features.

I hope with these case examples we were able to showcase the expertise we have here at Mayo Clinic. We would now like to bring us back to the original question in this presentation: Which test should I order when considering diagnostic exploratory testing?

Overall summary: Which test should I order?

Overall, whole genome sequencing is currently the most comprehensive test available on the market, however, there are lots of factors that can influence a decision to perform genome sequencing including insurance coverage and patient pay price, the patient's phenotype and differential diagnosis, the available specimen types for different types of analysis, and the turnaround time of any particular test. With all of that in mind, these are the diagnostic exploratory tests that we have here at Mayo Clinic. You can visualize here each test name as well as their pneumonic. There are also order codes for familial comparator samples for exome and genome. If you are interested in learning more, you can go to mayocliniclabs.com and find the respective landing pages for these tests. 

This table is a side-by-side comparison of the types of variants that we can detect with each of these different tests, highlighting that genome is the most comprehensive and can analyze all of the variant classes with the exception of mitochondrial CNVs which we anticipate will be available in a future state. Using this table, you can quickly see the pros and cons of each test, and the available sample types. I'm not going to be able to go through every single check box and X on this slide, but do feel free to pause here to get a nice comparison of these offerings.

Genetics ordering algorithm

This image illustrates a possible ordering algorithm, highlighting the different paths and testing modalities when considering a genetic condition. You can see we've tried to emphasize that many of these paths do eventually lead to genome sequencing, but this can be a first-tier test as can exome sequencing, chromosomal microarray, or panel testing. So it is just important to consider all of these different paths depending on your specific scenario for your patient. And we are always happy to help you select the most appropriate strategy for your patient if needed.

Mayo highlights

Finally, before we leave you today, just to highlight, Mayo Clinic Laboratories does go above and beyond with a lot of beneficial facets available after reporting, which is important to consider. We also have a vast amount of expertise in the diagnostic exploratory space.

Summary of diagnostic exploratory testing

Thank you so much for listening today. Mayo Clinic does have a very comprehensive suite of diagnostic exploratory testing which includes exome and genome sequencing and chromosomal microarray. The right test is really dependent on a lot of factors, however, to date, genome sequencing is the most comprehensive test available in the market.

Thank You

Questions?

Contact us: mcleducation@mayo.edu

Image credit: Getty Images

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MCL Education

This post was developed by our Education and Technical Publications Team.