Next Generation Sequencing based methods in genetic disease diagnostics
Abstract
Background: Identification of a germline pathogenic variant that increases risk of getting diseases in a family is important for the clinical management of the family members. DNA sequencing is an important molecular diagnostic technology that determines the order of nucleotides in an individual's genetic code. Next generation sequencing (NGS) technologies in DNA sequencing has revolutionized the research and diagnostics within the field of genetic disease, providing the opportunity to perform comprehensive genetic testing of large gene sets and to discover new causative genes. Targeted sequencing, whole exome sequencing, and whole genome sequencing are now widely employed by clinical laboratories using NGS, considering the benefits and difficulties of each technique. The project focuses on the study and use of these NGS technologies and developing bioinformatics analysis strategies for the same, suitable for usages in clinical diagnosis of hereditary diseases with focus on hereditary cancer.
Results: Studies I and III aimed to identify genetic variants that are associated with an increased risk of cancer, specifically endometrial and colorectal cancer respectively. Study I used gene panel sequencing to screen 22 genes involved in the mismatch repair pathway in 199 unselected endometrial cancer patients. Study III performed whole exome sequencing on 48 patients suspected of familial colorectal cancer. Bioinformatic pipelines were used to identify and classify variants, where use of multiple in silico tools improved the accuracy of predictions. Study I identified 22 potential pathogenic variants that may be associated with an increased risk of endometrial cancer, and Study III identified 26 germline variants in genes known for their association with colorectal cancer, as well as variants in other genes that may also contribute to an increased risk, hinting for a larger genetic spectrum of colorectal cancer, not limited to just mismatch repair genes. Study II demonstrated the development of a bioinformatic pipeline for copy number variation (CNV) detection in NGS data from diagnostic gene panels. With a sensitivity of 100% and specificity of 91%, the pipeline has been successful in detect CNVs in all control samples.
Conclusions: These studies (I & III) used gene target panel and exome sequencing to find the likely genetic cause for predisposition in several patients that participated in these studies. These studies also contribute to a larger understanding of the genetic spectrum of cancer. The bioinformatic pipeline (study II) has now been incorporated into routine practices, leading to expansion of the portfolio of genes for which CNV detection can be offered and demonstrated its diagnostic value by identifying CNVs in routine tests of patient samples. This has allowed for efficient and cost-effective CNV detection, which was previously limited by wet lab methods like MLPA. The outcomes of the whole project can help identify patients with inherited increased risk for cancers and other genetic disease, allowing for lifesaving surveillance.
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Article 1: Singh, Ashish Kumar; Talseth-Palmer, Bente Anita; McPhillips, Mary; Lavik, Liss Ane; Xavier, Alexandre; Drabløs, Finn; Sjursen, Wenche. Targeted sequencing of genes associated with the mismatch repair pathway in patients with endometrial cancer. PLOS ONE 2020 s. 1-19 https://doi.org/10.1371/journal.pone.0235613 This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0)Article 2: Singh, Ashish Kumar; Olsen, Maren Fridtjofsen; Lavik, Liss Ane; Vold, Trine; Drabløs, Finn; Sjursen, Wenche. Detecting copy number variation in next generation sequencing data from diagnostic gene panels. BMC Medical Genomics 2021 ;Volum 14.(1) s. 1-12 https://doi.org/10.1186/s12920-021-01059-x This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0)
Article 3: Singh, Ashish Kumar; Talseth-Palmer, Bente Anita; Xavier, Alexandre; Scott, Rodney J.; Drabløs, Finn Sverre; Sjursen, Wenche. Detection of germline variants with pathogenic potential in 48 patients with familial colorectal cancer by using whole exome sequencing. - The final published verwsion is available in BMC Medical Genomics 2023 ;Volum 16.(1) https://doi.org/10.1186/s12920-023-01562-3 This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0)