Next Generation Sequencing in Molecular Diagnosis of Lynch Syndrome – a Pilot Study Using New Stratification Criteria

Abstract

The development of the new technologies such as the next-generation sequencing (NGS) makes more accessible the diagnosis of genetically heterogeneous diseases such as Lynch syndrome (LS). LS is one of the most common hereditary form of colorectal cancer. This autosomal dominant inherited disorder is caused by deleterious germline mutations in one of the mismatch repair (MMR) genes – MLH1, MSH2, MSH6 or PMS2, or the deletion in the EPCAM gene. These mutations eventually result in microsatellite instability (MSI), which can be easily tested in tumor tissue. According to the actual recommendations, all patients with CRC that are suspect to have LS, should be offered the MSI testing. When the MSI is positive, these patients should be recommended to genetic counseling. Here we report a pilot study about the application of NGS in the LS diagnosis in patients considered to have sporadic colorectal cancer. The inclusion criteria for the NGS testing were MSI positivity, BRAF V600E and MHL1 methylation negativity. We have used 5 gene amplicon based massive parallel sequencing on MiSeq platform. In one patient, we have identified a new pathogenic mutation in the exon 4 of the MSH6 gene that was previously not described in ClinVar, Human Gene Mutation Database, Ensembl and InSight databases. This mutation was confirmed by the Sanger method. We have shown that the implementation of new criteria for colorectal patients screening are important in clinical praxis and the NGS gene panel testing is suitable for routine laboratory settings.

MSH2 Overexpression Due to an Unclassified Variant in 3’-Untranslated Region in a Patient with Colon Cancer

“…LS is an autosomal dominant condition caused by a defect in one of the MMR genes and is characterized by a high lifetime risk of tumor development, especially colorectal cancer (20-70%), endometrial cancer (15-70%), and other extracolonic tumors (15%) [7]. The molecular characterization of LS patients relies on the identification of point mutations and large rearrangements in the coding regions of the MMR genes, MLH1, MSH2, PMS2, and MSH6 [8][9][10][11][12][13].…”

A Scoring Model and Protocol to Adapt Universal Screening for Lynch Syndrome to Identify Germline Pathogenic Variants by Next Generation Sequencing from Colorectal Cancer Patients and Cascade Screening

“…Second, tumors are screened for signs of MSI or absence of MMR proteins using IHC. If these findings are conclusive, they are supplemented in a third step by NGS testing for pathological variants [ 17 , 32 , 33 ] ( Figure 1 ). Recently, there have been several attempts to shed light on which clinical predictors are most likely to be useful.…”

Diagnostics of Mutations in MMR/EPCAM Genes and Their Role in the Treatment and Care of Patients with Lynch Syndrome

“…With the NGS method, it is possible to sequence the whole genome (WGS, whole-genome sequencing), whole exome (WES, whole-exome sequencing), or to perform targeted gene sequencing. The main advantage of NGS is the ability to detect single nucleotide variations (SNVs) or small insertions/deletions in several genes simultaneously [ 7 , 86 , 87 ]. Useful features include short time of analysis, detection of meager input of nucleic acids [ 86 ], and sensitivity and specificity reaching 99.9% for both parameters [ 77 ].…”

“…The method consists of 5-6 steps and was described in detail for the first time by Schouten et al [90]. In LS diagnostics, commercial targeted gene panels, including exons and exon-intron regions of MMR/EPCAM genes, are available (e.g., HNPCC MASTR Plus, Agilent) [86]. However, it is also possible to design panels depending on needs.…”

“…A summary of the advantages and limitations of molecular applications is presented in Table 6. [86,88], detection of SNVs and small insertions/deletions [94,95] advanced bioinformatics systems and large data storage potential [96,97], filtering and data interpretation (various variants can be found when a large number or whole genes are sequenced) [94,97], issues with detecting structural rearrangements or copy number variations (CNVs) [95] Multiplex Ligation-dependent Probe Amplification (MLPA) wide diagnostic applications-copy numbers, point mutations detection, methylation profiling, also detected simultaneously, washing unbounded probes are not necessary, a simple and cost-effective method, easy analysis of the results [91,92] does not detect balanced mutations, like balanced translocations or inversions (detects only ones which affect the probe binding sequence), probes can be designed only for known mutations-impossible to detect an unknown mutation, the heterozygous deletions analysis is reliable when tumor cells constitute 20-30% of the sample, heterozygous duplication-about 40% [91,92], does not provide precise deletion/insertion characteristics' [98] High-Resolution Melting (HRM) simple after proper optimization, fast, high-throughput, software supporting optimization available, relatively simple and not-expensive equipment needed [98,99] detected variants not characterized, further characterization with another method, e.g., sequencing needed [98,99] Sanger sequencing the gold standard, mainly for detecting point mutations, high quality reads [98] not cost-effective when a large number of samples and long sequences are analyzed, technically demanding method [98] Single-Strand Conformation Polymorphism (SSCP) detection of point mutations, deletions, and insertions, detection of unknown variants, simple and quite fast method [81] low sensitivity and repeatability, amplicons not longer than 200-300 bp, detected variants not characterized, further characterization with another method, e.g., sequencing needed [80] Table 6. Cont.…”