Leading with quality: full PMS2 testing

Jul 12 · 3 min read

Originally published on Invitae.com on October 28, 2016

Invitae’s mission is to make high-quality genetic testing accessible to everyone who needs it. This is the first installment of the Leading with Quality series, which walks you through the many stringent processes and standards we use to provide you with the answers you need — accurately and reliably.

This week, we’re focusing on our approach to detecting mutations in the difficult gene PMS2.


Analyzing PMS2 is complicated by a pseudogene that is downstream of PMS2, called PMS2CL. The sequences of PMS2 (exons 12–15) and PMS2CL (exons 3–6) are almost exactly the same, making it very difficult to tell if a variant is in the gene or the pseudogene using conventional methods (e.g., Sanger sequencing, microarray, NGS).

PMS2 mutations are being found to be the cause of Lynch syndrome in an increasingly large group of patients who would ordinarily not have been tested for mismatch repair defects because their family history failed to meet Amsterdam or Bethesda criteria. We recognize the importance of identifying these variants correctly to avoid missing or incorrectly diagnosing Lynch syndrome cases.

What methods are available that distinguish between variants in PMS2 and PMS2CL?

The current gold standard (Vaughn et al. 2011) for differentiating between variants in PMS2 and PMS2CL is long range PCR (LR-PCR) followed by traditional Sanger sequencing for sequence variants and MLPA followed by LR-PCR and Sanger sequencing to identify deletions or duplications. Not every lab uses methods that can detect true positive PMS2 mutations reliably; those that do not use such methods have exclusions and limitations in their assay.

How does Invitae do this?

Invitae’s approach to evaluating exons 12–15 of PMS2 is a two-step process for read-through variants and a three-step process for deletions and duplications.

Sequence (read through) variants in exons 12–15

  • Step 1: NGS screen to identify non-benign variants
  • Step 2: Long range PCR and Sanger sequencing


  • Step 1: NGS screen to identify non-benign variants
  • Step 2: Confirm NGS data with MPLA
  • Step 3: Long range PCR and Sanger sequencing

A summary of this methodology and preliminary data was recently presented at CGA and ASHG.

Why is this important?

Between August 2015 and June 2016, Invitae testing uncovered 265 pathogenic and likely pathogenic variants in PMS2 and PMS2CL. Most (210) were in the pseudogene (PMS2CL), 44 were in PMS2, and 1 could not be disambiguated.* This small dataset, collected from our first 9 months of full PMS2 analysis, shows the importance of a gold standard method for testing and then confirming variants.

Of the pathogenic and likely pathogenic calls in PMS2, 60% met testing guidelines — most due to abnormal PMS2 staining on colon tumors and/or young age of colorectal cancer. One patient had a family history that met Amsterdam criteria. Approximately 40% were found “incidentally” as a result of panel testing for other indications such as family history of breast and other cancers. In addition, in cases when MLPA was not informative for deletions and duplications, Invitae was able to disambiguate 25% more cases using NGS data.

Having reliable mutation detection for PMS2 is clearly important not only in Lynch syndrome families, but in all testing for hereditary cancer predisposition.

Test your testing provider

With more and more PMS2 pathogenic variants being found, we encourage you to test your testing provider by asking:

  • Is testing for PMS2 exons 12–15 included in the assay? If so, what are the methods for distinguishing between PMS2 and PMS2CL mutations (e.g., LR-PCR and MLPA)?
  • How were the methods validated?
  • How often is the lab unable to distinguish between PMS2 and PMS2CL mutations and, when that happens, is their report transparent in stating the test failed to make that distinction?

If you don’t have the opportunity to discuss your testing provider’s methods with them, we also highly recommend looking at their website and reading the fine print about PMS2!

If you have any questions about Invitae’s approach to testing and confirmation — or about our dedication to high quality testing in general — please don’t hesitate to contact Client Services. We look forward to hearing from you.

*For the one variant that could not be disambiguated, Invitae’s report language read: “This is a deletion of either exon 14 of PMS2 or exon 5 of PMSCL. The unique sequence differences that can usually distinguish between these two locations are not present.”


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Invitae’s mission is to bring genetics to mainstream medicine to improve healthcare for billions.


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Invitae Corporation is a genetics company committed to providing genetic information to help inform healthcare decisions throughout all stages of life.


Invitae’s mission is to bring genetics to mainstream medicine to improve healthcare for billions.