Detecting clinically actionable variants in the 3' exons of PMS2 via a reflex workflow based on equivalent hybrid capture of the gene and its pseudogene

Abstract

Background: Hereditary cancer screening (HCS) for germline variants in the 3' exons of PMS2, a mismatch repair gene implicated in Lynch syndrome, is technically challenging due to homology with its pseudogene PMS2CL. Sequences of PMS2 and PMS2CL are so similar that next-generation sequencing (NGS) of short fragments-common practice in multigene HCS panels-may identify the presence of a variant but fail to disambiguate whether its origin is the gene or the pseudogene. Molecular approaches utilizing longer DNA fragments, such as long-range PCR (LR-PCR), can definitively localize variants in PMS2, yet applying such testing to all samples can have logistical and economic drawbacks.

Methods: To address these drawbacks, we propose and characterize a reflex workflow for variant discovery in the 3' exons of PMS2. We cataloged the natural variation in PMS2 and PMS2CL in 707 samples and designed hybrid-capture probes to enrich the gene and pseudogene with equal efficiency. For PMS2 exon 11, NGS reads were aligned, filtered using gene-specific variants, and subject to standard diploid variant calling. For PMS2 exons 12-15, the NGS reads were permissively aligned to PMS2, and variant calling was performed with the expectation of observing four alleles (i.e., tetraploid calling). In this reflex workflow, short-read NGS identifies potentially reportable variants that are then subject to disambiguation via LR-PCR-based testing.

Results: Applying short-read NGS screening to 299 HCS samples and cell lines demonstrated >99% analytical sensitivity and >99% analytical specificity for single-nucleotide variants (SNVs) and short insertions and deletions (indels), as well as >96% analytical sensitivity and >99% analytical specificity for copy-number variants. Importantly, 92% of samples had resolved genotypes from short-read NGS alone, with the remaining 8% requiring LR-PCR reflex.

Conclusion: Our reflex workflow mitigates the challenges of screening in PMS2 and serves as a guide for clinical laboratories performing multigene HCS. To facilitate future exploration and testing of PMS2 variants, we share the raw and processed LR-PCR data from commercially available cell lines, as well as variant frequencies from a diverse patient cohort.

Keywords: Hereditary cancer screening; Lynch syndrome; PMS2; PMS2CL; Reflex testing.

Fig. 1

LR-PCR strategy for building a dataset of natural genetic variation in PMS2 and PMS2CL. a Short-reads from NGS hybrid-capture data that originate from the gene (blue) and pseudogene (red) align to both the gene and pseudogene due to high homology. b, c Using LR-PCR that is specific to the gene or pseudogene followed by fragmentation and barcoding (b), the resulting short NGS reads can be assigned to the gene or pseudogene (c). d Percent identity between the gene and pseudogene for PMS2 exons 11–15 based on the hg19 reference genome (gray) and after accounting for natural genetic variation obtained from LR-PCR samples (black)

Fig. 2

Reflex workflow for variant identification in the last exons of PMS2. a Overview of sequencing and analysis workflow for the last five exons of PMS2. Colored nodes correspond to boxes in (b). b Details corresponding to workflow steps in (a); the details of each box are described in Methods and Results. “No report” means the variant does not appear on patient reports. “Reflex” means the sample is sent for LR-PCR-based disambiguation to determine if the variant is localized to the gene or pseudogene

Fig. 3

Hybrid-capture and LR-PCR are concordant for SNVs and indels. a Hypothetical examples to describe the concordance table for comparison of hybrid capture and LR-PCR data. All examples assume the reference base is A and the alternate (“alt”) base is T. (i) Example of a true positive (dark blue) where an alt allele is present in PMS2CL. (ii) Example of a permissible dosage error (light blue), where PMS2CL is homozygous for the alt allele but hybrid capture only calls one alt allele instead of two. (iii) Example of a false positive (light orange), where only hybrid capture detected an alt allele. (iv) Example of a false negative (dark orange), where an alt allele in PMS2CL was missed by hybrid capture. Shaded matrix on the right indicates cells that represent true positives, permissible dosage errors, false positives, and false negatives. Numbers on axes denote the total number of alt alleles in either the hybrid capture data or the PMS2/PMS2CL LR-PCR data. b Diploid SNV and indel concordance for exon 11 of PMS2. Numbers on axes denote the number of alt alleles where 0 is equivalent to 0/0, 1 is equivalent to 0/1, and 2 is equivalent to 1/1. 95% confidence intervals in brackets. c Four-copy SNV and indel concordance for exons 12–15 of PMS2/PMS2CL, as explained in (a)

Fig. 4

Simulated indels increase confidence in indel sensitivity. a Schematic of simulating a tetraploid indel by combining sequencing data from two diploid samples. b Results of tetraploid indel simulations in the same format as Fig. 3a

Fig. 5

Hybrid capture, LR-PCR, and MLPA are concordant for CNVs. a All CNVs called in the hybrid capture data and corresponding orthogonal confirmation data. b Hybrid capture data for the patient sample with an exon 13–14 deletion depicts copy-number estimates across the locus (bins). Gray regions denote the last four exons of PMS2. White regions denote introns. Yellow box indicates region of the CNV call. c MLPA data for the exon 13–14 deletion patient sample. PMS2-specific (solid blue), PMS2CL-specific (solid red), and PMS2/PMS2CL degenerate MLPA probes (blue and red stripes) show the deletion in exons 13–14 of PMS2CL. d LR-PCR data for the exon 13–14 deletion sample depicting copy number estimates across the locus (bins) for PMS2 (blue, top) and PMS2CL (red, bottom). Gray regions depict exons 11–15 of PMS2 and white regions depict introns as in (b)