Nicole Hoppman, Ph.D., is a Consultant in Laboratory Genetics and Genomics for the Department of Laboratory Medicine and Pathology at Mayo Clinic in Rochester, Minnesota. She holds the academic rank of Assistant Professor of Laboratory Medicine and Pathology.
Contact us: MMLHotTopics@mayo.edu.
Hi, I’m Matt Binnicker, the Director of Clinical Virology and Vice Chair of Practice in the Department of Laboratory Medicine and Pathology at Mayo Clinic. Did you know that rearrangements in certain regions of human chromosomes can occur in phenotypically normal individuals, but they can also result in intellectual disabilities, physical malformations, and a wide variety of cancers? In this month’s "Hot Topic," my colleague Dr. Nicole Hoppman will discuss an exciting technology, called "mate-pair sequencing," which is the first clinically available test that can characterize almost any chromosomal rearrangement, helping to establish pathogenicity, and, in a neoplastic setting, assist in diagnosis, prognosis, and identification of optimal therapeutic options. I hope you enjoy this month’s "Hot Topic," and I want to personally thank you for allowing Mayo Clinic the opportunity to be a partner in your patients’ health care.
Thank you so much for the introduction. I have no disclosures.
As you view this presentation, consider the following important points regarding testing. How is the testing going to be used in your practice? When should the tests be used? And how will results impact patient management?
Introduction: Chromosomes and Cytogenetics
Today, I will be discussing a novel group of clinical assays that utilize mate-pair-sequencing technology to answer specific clinical questions. First, however, I will give some background information to help demonstrate the necessity and utility of this type of testing. The image shown here is a karyotype of a female obtained by performing G-banded chromosome studies.
Chromosome studies have been one of the “gold standards” of cytogenetic testing for several decades. While chromosome studies provide a snapshot of a patient’s entire genome, they have low resolution and cannot provide information regarding the gene content of genomic rearrangements and copy number variants.
Chromosome studies can detect many different types of chromosome abnormalities, including balanced rearrangements such as inversions and translocations. Balanced chromosome rearrangements, such as the 4;18 translocation shown in this karyotype, are often observed in a congenital, or germline, setting in 1 of 2 scenarios. First, they can be detected in phenotypically normal individuals, usually in situations where there is a history of miscarriages, fertility problems, or phenotypically abnormal offspring who have an unbalanced version of the rearrangement. However, there are times in which they are observed in phenotypically abnormal individuals. In the latter situation, it is unclear whether or not the rearrangement is causing or contributing to the abnormal phenotype in the patient. The next step for these patients involves testing of the parents to determine whether the rearrangement was inherited from a parent who does not share phenotypic features with the patient; however, even if found to be de novo, or not inherited, the clinical significance remains uncertain.
Chromosome studies also frequently reveal balanced chromosome rearrangements in neoplastic settings, both in hematologic malignancies such as acute leukemias as well as solid tumors. In a cancer setting, chromosome rearrangements such as the 5;6 translocation shown here can have one of many effects, such as the creation of oncogenic fusion genes, disruption of a tumor-suppressor gene, or activation of an oncogene via position effects. These rearrangements, depending on the genes involved, can greatly impact diagnosis, prognosis, and even choice of therapeutic options for the patient.
However, the bottom line is that chromosome studies are not able to identify what genes are at or near the breakpoints of a rearrangement. Some rearrangements, such as the 9;22 translocation typically seen in patients with chronic myelogenous leukemia, are “classic,” and their gene content can be inferred and/or confirmed by other techniques such as fluorescence in situ hybridization (FISH) or RT-PCR. For the majority of rearrangements observed, however, there was not, until recently, a clinical test available to further characterize these chromosome rearrangements in order to determine their clinical significance. Mate-pair sequencing is a technology that has recently made this possible.
Mate-Pair Sequencing (MPseq)
Mate-pair sequencing takes advantage of a specialized library preparation technique followed by whole-genome, paired-end next-generation sequencing. The input DNA is large (3 to 5 kilobases) as opposed to the typical 2 to 500 base-pairs size that most NGS applications require. This increased size allows for efficient detection of structural variation in the genome at lower sequencing depth (and therefore cost) than other NGS methods.
This diagram gives a high-level overview of the mate-pair-sequencing process. First, genomic DNA is sheared to approximately 3 kilobase fragments. The ends of these fragments are represented by the red and blue bars, and the ends of these fragments are biotinylated, represented by the letter B. These large fragments of DNA are circularized, bringing the ends of the 3 kilobase fragment of DNA together. This circular fragment is then sheared, and a streptavidin capture is performed so that mostly only the pieces of DNA representing the junctions where the ends of our large pieces of input DNA were joined are captured and sequenced. Sequencing is performed in a paired-end manner, meaning both ends of the fragments are sequenced. These reads are then mapped to the human genome, where any two ends of a fragment would be expected, in the absence of any type of structural variation, to map to the same chromosome approximately 3 kilobases apart in a specific orientation. This is what we call “concordant fragments.”
However, when structural variation is present, we observe what are called discordant fragments. This means that the paired ends map either to different chromosomes, such as in this example where one end maps to chromosome 3 and the other to chromosome 7, representing a translocation with the breakpoint located somewhere between these two reads, or where the reads map to the same chromosome but in unexpected locations and/or orientations, indicative of an inversion. Complex rearrangements involving the same or multiple chromosomes can also be detected. It is possible to tease out, by looking at the mapping patterns and genomic locations, what the rearrangement is and what genes are affected. Mate pairs can typically resolve a breakpoint to within 2 to 3 kilobases of a breakpoint, which is smaller than the size of most genes, and if necessary, other methods such as Sanger sequencing can be utilized to resolve the breakpoint to the single nucleotide level.
3 Clinical Assays Live to Date
Mayo clinic recently went live with three clinical assays using this technology:
- MTRBL, which is a mate-pair assay for congenital chromosome abnormalities that is performed on peripheral blood.
- MTRBM, which is for hematologic neoplasms (leukemias and lymphomas) and is performed on bone marrow or blood specimens.
- And MTRTI, which is for solid-tumor specimens and can be performed on either fresh or frozen tissue or cell cultures. At this time, formalin-fixed, paraffin-embedded (FFPE) specimens cannot be accepted for mate-pair assays.
Mate-Pair Clinical Assays
All three mate-pair assays are follow-up or add-on assays that can be utilized when a chromosome rearrangement by FISH or chromosomes is identified, and there is possible utility in characterizing the rearrangement further. Although the sequencing is whole-genome, the analysis that is performed is targeted and depends on the rearrangement of interest. Therefore, a copy of previous test results is required in order to determine the target rearrangement that is to be analyzed. These assays are not whole-genome analyses, replacements for any other tests offered by Mayo Clinic, or standalone tests. At this time, mate-pair assays are not useful for monitoring minimal residual disease or detecting single nucleotide variants.
Possible Scenario: MTRBM
The following illustrates a scenario in which mate-pair testing, specifically the MTRBM test, could not only be useful but also provide clinically actionable information. In this case, we have a 10-year-old female with a history of B-cell acute lymphoblastic leukemia who is currently relapsing. FISH studies with a probe for the ETV6 gene had indicated disruption of ETV6, possibly the result of a 12;15 translocation; however, the partner gene was not obvious, making the significance of this rearrangement unclear as ETV6 disruption is common in a wide variety of cancers. But the specific prognosis associated with disruption of this gene depends on the partner gene.
Bone Marrow: Chromosome Studies
This is an example of the karyotype obtained by chromosome studies for this patient, showing a hyperdiploid clone, but the absence of obvious abnormalities of chromosomes 12 and 15 mean that this rearrangement, suggested by FISH studies that is shown on the next slide, is “cryptic” or below the resolution of a chromosome study.
These are the interphase FISH images for probes specific for the ETV6 gene, located on chromosome 12, where the yellow signals indicate intact ETV6 genes, and the red and green signals represent disrupted ends of the gene, indicating a rearrangement.
Since chromosome studies did not identify an abnormality of chromosome 12, metaphase FISH was also performed, demonstrating two normal chromosome 12’s with intact ETV6 as well as one abnormal chromosome 12 with the 3’ end of the ETV6 gene, and two abnormal chromosome 15’s, both with the 5’ end of ETV6. This indicates a likely 12;15 translocation.
As mentioned earlier, ETV6 disruption occurs in many tumor types, both solid tumors as well as hematologic malignancies, and ETV6 has multiple possible partner genes. ETV6 is interesting because it also has multiple mechanisms when disrupted, including constitutive activation of its partner gene via formation of a chimeric gene, loss of function, activation of nearby oncogenes via positional effects, etc. Without information regarding the partner gene, however, the clinical significance of this disruption is uncertain for this patient. In addition, our laboratory does not have any clinically validated probes for genes on chromosome 15 that could potentially be the partner gene involved in this rearrangement.
This is a perfect example of where mate-pair testing can be utilized. In this case, mate pair was performed and identified the 12;15 rearrangement, shown by the purple line between chromosomes 12 and 15 in this whole-genome view.
If we zoom in at what we call the junction plot for the 12;15 rearrangement, we see that the breakpoint on chromosome 12, which is on top, indeed lies within the ETV6 gene, while the breakpoint on chromosome 15 resides within the NTRK3 gene.
Targetable Kinase Gene Fusions in B-ALL
Interestingly, as you see here in this figure from Reshmi, et al., that NTRK3 is rare but known targetable tyrosine kinase in B-cell ALL.
In this particular case, this 12;15 translocation results in a chimeric gene consisting of exons 1–4 of ETV6 and exons 15–20 (including the tyrosine kinase domain) of NTRK3, likely resulting in up-regulation of the tyrosine kinase domain of NTRK3. This particular chimeric gene is very rare and has been only reported in a few cases of B-cell ALL as well as some other tumor types. Most importantly, however, it is targetable using tyrosine kinase inhibitors such as crizotinib. In this case, the mate-pair result led to additional treatment options for this patient.
Possible Scenario: MTRTI
The mate-pair test for solid tumors is very similar to that for hematologic malignancies, so in the interest of time, here is a very brief example scenario: Tumor cells from an 18-year-old woman are found to have an acquired inversion of chromosome X as a sole abnormality. Mate-pair, whole-genome sequencing is requested to determine the genes at/near the breakpoints in order to subtype the neoplasm, provide prognostic predictions, and/or possibly identify therapeutic targets.
Possible Scenario: MTRBL
And, finally, here is a brief scenario for the congenital test: A 6-month-old boy with a congenital malformation, developmental delay, and intellectual disability is found to have a 1;19 translocation by chromosome analysis. Neither of his parents has this same translocation. Mate-pair sequencing can be used to determine what genes are disrupted by the translocation in order to provide possible diagnostic information. The important difference in a congenital setting is that is it preferable to rule out the possibility of a rearrangement being inherited from a phenotypically normal parent before mate-pair sequencing is performed.
In summary, mate-pair sequencing is a novel clinical assay used to characterize chromosome rearrangements observed by other clinical testing such as chromosome studies and/or FISH. Mayo Clinic’s three assays allow us to fill gaps in clinical testing, allowing advances in diagnosis, prognosis, and in some oncology cases, possibly opening up new therapy options that would not have been available without this test.