Development of Method in Screening Individual with Lynch Syndrome

By: Ryia Illani Mohd Yunos (ryia.yunos@ppukm.ukm.edu.my)

Introduction

ryiaColorectal cancer is generally classified into two main categories – sporadic and familial. About 5 to 10% of colorectal cancer cases are familial and it is estimated that about 1 to 5 percent of cases are due to mutations in highly penetrant single gene. Lynch syndrome is associated with mutations in mismatch repair (MMR) genes (MLH1, MSH2, MSH6, and PMS2) and individuals who have mutations in any of these genes have 20 to 65% lifetime risk of colorectal cancer, compared with a lifetime risk among the general population of approximately 5%. Because its inheritance is autosomal dominant, close biological relatives are also at high risk. A systematic review conducted by Kriza and colleagues in 2013 showed that long-term costs for colorectal cancer can go up to $50,175 per patient (2008 values). Hence, prevention as well as early detection of CRC may lead to better health outcomes and considerable savings in treatment costs. Progress in reducing colorectal cancer morbidity can be accelerated by screening individuals who has high risk of getting CRC and discover the diseases at its early stage. Therefore, the aim of this study is to develop a rapid, cost effective and sensitive method of screening Lynch syndrome.

Method

image001Ion Ampliseqâ„¢ Custom Panel with four MMR genes associated with Lynch syndrome (MLH1, MLH2, MSH6 and PSM2) and two genes which is not categorized as MMR genes (EPCAM and BRAF) was designed and simultaneously sequence on Ion Torrent PGMâ„¢ sequencer. EPCAM is located adjacent to MSH2 and mutation of the 3′ end of EPCAM can affect MSH2 gene expression whereby EPCAM 3′ deletion can lead to inactivation of MSH2 gene (Kempers et al., 2011). Therefore, EPCAM should also be included in this panel for screening process of hereditary CRC. BRAF gene mutation is predominantly associated with sporadic CRC (Domingo et al., 2004). Testing of this gene will essentially rule out diagnosis of hereditary CRC.

Outcome

Sequencing of these six genes in the 19 samples generated a mean of 664, 260 reads per patient. On average, 92% of these reads mapped to the targeted region (MLH1, MSH2, MSH6, PMS2, EPCAM and BRAF), with 98% uniformity across all target. The percentage of reads mapped to the target is rather a bit low due to paralogous gene to PMS2 which is PMS2CL. We used three different pipeline to analyze the data: BWA-GATK, Samtools-Bowtie and TMAP-TVC.

BowtieFrom these analyses, 58 overlapped variants were identified and chosen to be validated via MassArray and Sanger Sequencing in 19 samples. Upon completion of the validation, 93.4% of the identified variants were in concordant with targeted sequencing. A pathogenic variant in MSH2 gene was identified in a 44 years old Dukes’ D CRC patient. The Ion Torrent PGM clearly identified a single base pair C to T substitution in MSH2 gene with a variant frequency of 52%. This was confirmed by Mass Array. Genetic counseling is recommended for this patient and for other at risk family members. Since we have documented the presence of pathogenic variant in this patient, testing of at risk individuals in the family is possible.

HCTThe sensitivity of the Ion torrent PGM for mutation detection was determined by sequencing serially diluted DNA from two human cancer cell lines; HCT 116 and LN18. DNA from HCT 116 was diluted in DNA from LN18 resulting in 25% and 5% dilution respectively. HCT 116 is a cell line with MLH1 p.S252X while LN18 was negative for this mutation. From the sequencing, we manage to identify the mutation with the variant frequency of about 25-30% and 10-13% for 25% dilution and 5% dilution, respectively. We have validated the mutation detected in our sensitivity sample using Sanger sequencing. However due to the limit of detection by Sanger sequencing we could not identify as low as 5% of allele frequency.

Conclusions

  1. Multiplex PCR followed by NGS is useful for screening individual with Lynch Syndrome by detecting germline mutation in MMR genes.
  2. BRAF gene mutation is predominantly associated with sporadic CRC. Testing of this gene will essentially rule out diagnosis of hereditary CRC.
  3. We achieved 92% specificity and 93.4% accuracy of the developed Lynch syndrome panel.
  4. Sensitivity achieved was 13% allelic frequency.
  5. However, further investigation on method to detect InDels in MMR genes as well alterations in PMS2 (due to highly homology to PMS2CL) is warranted and this is currently our on-going project.

References

  • Domingo, E., Laiho, P., Ollikainen, M., Pinto, M., Wang, L., French, A. J., … Schwartz, S. (2004). BRAF screening as a low-cost effective strategy for simplifying HNPCC genetic testing. Journal of Medical Genetics, 41(9), 664–8. http://doi.org/10.1136/jmg.2004.020651
  • Kempers, M. J. E., Kuiper, R. P., Ockeloen, C. W., Chappuis, P. O., Hutter, P., Rahner, N., … Ligtenberg, M. J. L. (2011). Risk of colorectal and endometrial cancers in EPCAM deletion-positive Lynch syndrome: a cohort study. The Lancet. Oncology, 12(1), 49–55. http://doi.org/10.1016/S1470-2045(10)70265-5
  • Kriza, C., Emmert, M., Wahlster, P., Niederlaender, C., & Kolominsky-Rabas, P. (2013). Cost of Illness in Colorectal Cancer: An International Review. Pharmacoeconomics, 31(7), 577–588. http://doi.org/10.1007/s40273-013-0055-4