Dan Kvitek

­Full PMS2 testing at Invitae: We’ve got you covered

Dan Kvitek, R&D scientist, Invitae

Lynch syndrome, also known as hereditary nonpolyposis colorectal cancer, is an inherited predisposition to many types of cancer, including colon, endometrium, ovary, stomach, and urinary tract.1 Most cases of this important cancer syndrome are caused by genetic variants in the genes MLH1, MSH2, and MSH6, but 4 to 11 percent of cases are caused by variants in the gene PMS2.2,3,4

Although sequencing MLH1, MSH2, and MSH6 is relatively straightforward, sequencing the PMS2 gene is much more difficult. To analyze complex portions of this gene for variants correctly, one must give it special treatment, on either the lab or bioinformatics side—or on both.

This special treatment often requires lab techniques that are expensive and complex. That’s because it has a tricky neighbor in the human genome—a non-functional duplicate or “pseudogene” known as PMS2CL. Portions of PMS2CL are so similar to PMS2 that when you target PMS2, you end up sequencing both PMS2 and PMS2CL, making it difficult to distinguish the gene from the pseudogene. To make matters more difficult, a process called gene conversion can lead to PMS2CL DNA sequence occurring in PMS2, and vice-versa.5 These unavoidable challenges make it impossible to sequence PMS2 using the same methods used for other genes, like MLH1, MSH2, and MSH6, while maintaining our very high standards of quality and confidence.

Now, thanks to some innovative thinking, we have developed a method for full PMS2 sequencing and deletion/duplication analysis with the same high quality and the same low price you’ve come to expect from Invitae.

The Invitae PMS2 bioinformatics screen

It has been said that necessity is the mother of invention. At Invitae, we often interpret this as “technical challenges breed creative innovation.” Staying true to this mentality, we went out of our way to develop a method of sequencing PMS2 that is very high quality and that delivers both read-though variants and deletions/duplications that meet or exceed industry standards. We aimed to add this capability in a cost-effective manner, and we succeeded in adding full PMS2 to our test menu while maintaining the same low price.

Our new, highly accurate sequencing method covers both PMS2 and PMS2CL the same way that other genes are covered. The trick lies in how these genes are aligned to the reference genome. By eliminating PMS2CL from the reference sequence, we ensure that reads from both PMS2 and PMS2CL align only to PMS2. First we identify samples with variants, then we follow up with standard confirmation assays to disambiguate whether it is PMS2 or PMS2CL that contains the variant. Importantly, in the common event of gene conversion between PMS2 and PMS2CL, this confirmation method enables us to accurately call variants in PMS2, which have the potential to be pathogenic, against variants in PMS2CL, which are not pathogenic.

All positive findings are confirmed with alternate technologies. We’ve also validated our approach to full PMS2 coverage, using a reference set of 60 samples carrying 295 true positive and 34,926 true negative variants in PMS2 or PMS2CL, and demonstrated an accuracy, precision, analytical sensitivity, and specificity of 100 percent.

What this means for you

The Invitae PMS2 screen is a win-win for clinicians and patients. Our approach keeps the price of our cancer testing low and the turnaround time short. Even more importantly, it ensures a high quality, clinical-grade diagnostic test for Lynch syndrome. It’s also a great example of how we’re reinventing genetic testing to make it more affordable and accessible than ever before.

To learn more about Invitae’s Lynch syndrome offerings, please view our Lynch syndrome webpage. You can also learn more about our approach to PMS2 in our technical validation paper.


1 Lynch, HT, et al. Review of the Lynch syndrome: History, molecular genetics, screening, differential diagnosis, and medicolegal ramifications. Clinical Genetics. 2009; 76:1-18. doi: 10.1111/j.1399-0004.2009.01230.x.

2 Gill, S, et al. Isolated loss of PMS2 expression in colorectal cancers: frequency, patient age, and familial aggregation. Clinical Cancer Research. 2005; 11:6466–6471. doi: 10.1158/1078-0432.CCR-05-0661.

3 Halvarsson, B, et al. The added value of PMS2 immunostaining in the diagnosis of hereditary nonpolyposis colorectal cancer. Familial Cancer. 2006; 5:353–358. doi: 10.1007/s10689-006-0005-9.

4 Truninger, K, et al. Immunohistochemical analysis reveals high frequency of PMS2 defects in colorectal cancer. Gastroenterology. 2005; 128:1160–1171. doi: 10.1053/j.gastro.2005.01.056.

5 Hayward, BE, et al. Extensive gene conversion at the PMS2 DNA mismatch repair locus. Human Mutation. 2007; 28:424–30. doi: 10.1002/humu.20457.