A massive parallel sequencing workflow for diagnostic genetic testing of mismatch repair genes

Hansen, Maren Fridtjofsen; Neckmann, Ulrike; Lavik, Liss Anne S.; Vold, Trine; Gilde, Bodil; Toft, Ragnhild Karlgård; Sjursen, Wenche

doi:10.1002/mgg3.62

Cited by 13 publications

(15 citation statements)

References 36 publications

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“…Next-generation sequencing approaches permit the simultaneous analysis of multiple genes in a limited period of time. This multigene diagnostic approach has been already fruitfully applied in oncology [ 30 , 15 ], and its introduction in routine practice for the molecular characterization of probands of colorectal cancer syndromes is foreseen [ 31 ].…”

Section: Discussionmentioning

confidence: 99%

Next-generation sequencing for genetic testing of familial colorectal cancer syndromes

Simbolo¹,

Mafficini²,

Agostini

et al. 2015

Hered Cancer Clin Pract

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BackgroundGenetic screening in families with high risk to develop colorectal cancer (CRC) prevents incurable disease and permits personalized therapeutic and follow-up strategies. The advancement of next-generation sequencing (NGS) technologies has revolutionized the throughput of DNA sequencing.MethodsA series of 16 probands for either familial adenomatous polyposis (FAP; 8 cases) or hereditary nonpolyposis colorectal cancer (HNPCC; 8 cases) were investigated for intragenic mutations in five CRC familial syndromes-associated genes (APC, MUTYH, MLH1, MSH2, MSH6) applying both a custom multigene Ion AmpliSeq NGS panel and conventional Sanger sequencing.ResultsFourteen pathogenic variants were detected in 13/16 FAP/HNPCC probands (81.3 %); one FAP proband presented two co-existing pathogenic variants, one in APC and one in MUTYH. Thirteen of these 14 pathogenic variants were detected by both NGS and Sanger, while one MSH2 mutation (L280FfsX3) was identified only by Sanger sequencing. This is due to a limitation of the NGS approach in resolving sequences close or within homopolymeric stretches of DNA. To evaluate the performance of our NGS custom panel we assessed its capability to resolve the DNA sequences corresponding to 2225 pathogenic variants reported in the COSMIC database for APC, MUTYH, MLH1, MSH2, MSH6. Our NGS custom panel resolves the sequences where 2108 (94.7 %) of these variants occur. The remaining 117 mutations reside inside or in close proximity to homopolymer stretches; of these 27 (1.2 %) are imprecisely identified by the software but can be resolved by visual inspection of the region, while the remaining 90 variants (4.0 %) are blind spots. In summary, our custom panel would miss 4 % (90/2225) of pathogenic variants that would need a small set of Sanger sequencing reactions to be solved.ConclusionsThe multiplex NGS approach has the advantage of analyzing multiple genes in multiple samples simultaneously, requiring only a reduced number of Sanger sequences to resolve homopolymeric DNA regions not adequately assessed by NGS. The implementation of NGS approaches in routine diagnostics of familial CRC is cost-effective and significantly reduces diagnostic turnaround times.Electronic supplementary materialThe online version of this article (doi:10.1186/s13053-015-0039-9) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Next-generation sequencing for genetic testing of familial colorectal cancer syndromes

Simbolo¹,

Mafficini²,

Agostini

et al. 2015

Hered Cancer Clin Pract

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“…However, both mutations were classified as “Pathogenic” in the ClinVar database. Single heterozygous stop gain mutation of MSH6 c.3103C>T p.Arg1035Ter was previously described in a family with endometrial cancer and rectal cancer, and also the Lynch syndrome, while single frameshift heterozygous mutation of MSH6 c.3261dupC p.Phe1088LeufsTer was previously reported in multiple kinds of tumors, including the Lynch syndrome, colorectal cancer, prostate cancer, and endometrial cancer . In addition, c.3261dupC variant has been seen in the homozygous state in several individuals from a consanguineous family with CMMR‐D syndrome, and in this family, the carriers parents had not presented any tumor, but should participate the preventive program for Lynch syndrome .…”

Section: Discussionmentioning

confidence: 95%

Rare compound heterozygous mutations in gene MSH6 cause constitutive mismatch repair deficiency syndrome

Ling

Yang

Sun

et al. 2018

Clinical Case Reports

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Key Clinical MessageFew studies reported patients who harbored three kinds of primary tumors simultaneously. Here, we present a 9‐year‐old boy with colon carcinoma, brain medulloblastoma, and lymphoma. Genetic mutation detection was explored with next‐generation sequencing, and compound heterozygous mutations in gene MSH6 c.3103C>T p.Arg1035Ter and c.3261dupC p.Phe1088LeufsTer were discovered.

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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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A massive parallel sequencing workflow for diagnostic genetic testing of mismatch repair genes

Cited by 13 publications

References 36 publications

Next-generation sequencing for genetic testing of familial colorectal cancer syndromes

Next-generation sequencing for genetic testing of familial colorectal cancer syndromes

Rare compound heterozygous mutations in gene MSH6 cause constitutive mismatch repair deficiency syndrome

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

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