Clinical analysis of<i>PMS2</i>: mutation detection and avoidance of pseudogenes

Vaughn, Cecily P.; Robles, J.; Swensen, Jeffrey; Miller, Christine; Lyon, Elaine; Mao, Rong; Bayrak-Toydemir, Pınar; Samowitz, Wade S.

doi:10.1002/humu.21230

Cited by 58 publications

(87 citation statements)

References 25 publications

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“…Detection of pathogenic mutations using current methodologies is likely incomplete given the number of patient samples with isolated loss of PMS2 protein expression for which pathogenic mutations are not identified [Senter et al, 2008;Vaughn et al, 2010]. In those samples in which pathogenic mutations are detected, two studies showed that large deletions of one or more exons comprised 21% and 37% of PMS2 mutations [Senter et al, 2008;Vaughn et al, 2010].…”

Section: Introductionmentioning

confidence: 96%

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Avoidance of pseudogene interference in the detection of 3′ deletions in PMS2

et al. 2011

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Lynch syndrome is characterized by mutations in the mismatch repair genes MLH1, MSH2, MSH6, and PMS2. In PMS2, detection of mutations is confounded by numerous pseudogenes. Detection of 3' deletions is particularly complicated by the pseudogene PMS2CL, which has strong similarity to PMS2 exons 9 and 11-15, due to extensive gene conversion. A newly designed multiplex ligation-dependent probe amplification (MLPA) kit incorporates probes for variants found in both PMS2 and PMS2CL. This provides detection of deletions, but does not allow localization of deletions to the gene or pseudogene. To address this, we have developed a methodology incorporating reference samples with known copy numbers of variants, and paired MLPA results with sequencing of PMS2 and PMS2CL. We tested a subset of clinically indicated samples for which mutations were either unidentified or not fully characterized using existing methods. We identified eight unrelated patients with deletions encompassing exons 9-15, 11-15, 13-15, 14-15, and 15. By incorporating specific, characterized reference samples and sequencing the gene and pseudogene it is possible to identify deletions in this region of PMS2 and provide clinically relevant results. This methodology represents a significant advance in the diagnosis of patients with Lynch syndrome caused by PMS2 mutations.

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Section: Introductionmentioning

confidence: 96%

“…For sequencing, long-range amplification can be utilized to circumvent coamplification of pseudogenes [Clendenning et al, 2006;Vaughn et al, 2010]. For deletion/ duplication analysis by MLPA, it is necessary to locate probes over sequence unique to the gene.…”

Section: Introductionmentioning

confidence: 99%

Avoidance of pseudogene interference in the detection of 3′ deletions in PMS2

et al. 2011

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“…Regarding this point, our study suggests that PMS2, which does not contribute greatly to Lynch Syndrome [41], could be considered a good candidate for evaluation. Moreover, as the commonly known technical difficulties of analyzing PMS2 are slowly being overcome by new analysis strategies [41][42], critically needed evidence concerning the role of PMS2 can be expected. For instance, recently, ten Broeke et al reported a standardized incidence ratio of 3.8 for breast carcinomas, which led them to suggest adding mammography from age 40 years in PMS2 families with evident clusters of BC [43].…”

Section: Discussionmentioning

confidence: 99%

The transfer of multigene panel testing for hereditary breast and ovarian cancer to healthcare: What are the implications for the management of patients and families?

Lizard¹,

Eliade²,

Skrzypski

et al. 2016

JCO

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Until recently, the molecular diagnosis of hereditary breast and ovarian cancer (HBOC) was mostly based on BRCA1/2 testing. Next generation sequencing and the recent discovery of new genes involved in HBOC now permit the transfer of genomic capture targeting multiple candidate genes from research to clinical use. However, the implications for the management of patients and their families have not been extensively studied, in particular since some of these genes are not well-established cancer predisposing genes. We studied 583 consecutive patients from Burgundy

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“…The probe design of the TruSight Cancer Content suggests that the assay does not discriminate PMS2 from its 15 pseudogenes (36 ), and this would affect results interpretation. Previous studies have attempted to enrich for PMS2 using careful probe design (25,37,38 ); however, the high sequence homology has made absolute enrichment elusive. This emphasizes the need for careful probe design for accurate diagnosis, whether by NGS, Sanger sequencing, or alternative methods.…”

Section: Discussionmentioning

confidence: 99%

Feasibility of Low-Throughput Next Generation Sequencing for Germline DNA Screening

Sapari

Elahi

et al. 2014

Clinical Chemistry

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BACKGROUND Next generation sequencing (NGS) promises many benefits for clinical diagnostics. However, current barriers to its adoption include suboptimal amenability for low clinical throughputs and uncertainty over data accuracy and analytical procedures. We assessed the feasibility and performance of low-throughput NGS for detecting germline mutations for Lynch syndrome (LS). METHODS Sequencing depth, time, and cost of 6 formats on the MiSeq and Personal Genome Machine platforms at 1–12 samples/run were calculated. Analytical performance was assessed from 3 runs of 3 DNA samples annotated for 7500 nucleotides by BeadChip arrays. The clinical performance of low-throughput NGS and 9 analytical processes were assessed through blinded analysis of DNA samples from 12 LS cases confirmed by Sanger sequencing, and 3 control cases. RESULTS The feasibility analysis revealed different formats were optimal at different throughputs. Detection was reproducible for 2619/2635 (99.39%) replicate variants, and sensitivity and specificity to array annotation were 99.42% and 99.99% respectively. Eleven of 16 inconsistently detected variants could be specifically identified by having allele frequencies ≤0.15, strand biases >−35, or genotype quality scores ≤80. Positive selection for variants in the Human Genome Mutation Database (colorectal cancer, nonpolyposis) and variants with ≤5% frequency in the Asian population gave the best clinical performance (92% sensitivity, 67% specificity). CONCLUSIONS Low-throughput NGS can be a cost-efficient and reliable approach for screening germline variants; however, its clinical utility is subject to the quality of annotation of clinically relevant variants.

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Clinical analysis ofPMS2: mutation detection and avoidance of pseudogenes

Cited by 58 publications

References 25 publications

Avoidance of pseudogene interference in the detection of 3′ deletions in PMS2

Avoidance of pseudogene interference in the detection of 3′ deletions in PMS2

The transfer of multigene panel testing for hereditary breast and ovarian cancer to healthcare: What are the implications for the management of patients and families?

Feasibility of Low-Throughput Next Generation Sequencing for Germline DNA Screening

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