Title: | BRCA Testing by Single-Molecule Molecular Inversion Probes |
Author(s): | Neveling, K. ; Mensenkamp, A.R. ; Derks, R; Kwint, M.P. ; Ouchene, H.; Steehouwer, M. ; Lier, L.A. van; Bosgoed, E.A.J.; Rikken, A.; Tychon, M.W.J.; Zafeiropoulou, D.; Castelein, S.; Hehir-Kwa, J.Y. ; Thung, G.W.; Hofste, T.; Lelieveld, S.H.; Bertens, S.M.; Adan, I.B.; Eijkelenboom, A.; Tops, B.B.J.; Yntema, H.G. ; Stokowy, T.; Knappskog, P.M.; Hoberg-Vetti, H.; Steen, V.M.; Boyle, E.; Martin, B.; Ligtenberg, M.J.L. ; Shendure, J.; Nelen, M.R. ; Hoischen, A. |
Publication year: | 2017 |
Source: | Clinical Chemistry, vol. 63, iss. 2, (2017), pp. 503-512 |
ISSN: | 0009-9147 |
DOI: | https://doi.org/10.1373/clinchem.2016.263897 |
Publication type: | Article / Letter to editor |
Please use this identifier to cite or link to this item : https://hdl.handle.net/2066/169902 ![]() |
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Subject: | Radboudumc 0: Other Research RIHS: Radboud Institute for Health Sciences Radboudumc 12: Sensory disorders DCMN: Donders Center for Medical Neuroscience Radboudumc 14: Tumours of the digestive tract RIMLS: Radboud Institute for Molecular Life Sciences Radboudumc 4: lnfectious Diseases and Global Health RIMLS: Radboud Institute for Molecular Life Sciences Radboudumc 7: Neurodevelopmental disorders DCMN: Donders Center for Medical Neuroscience Radboudumc 7: Neurodevelopmental disorders RIMLS: Radboud Institute for Molecular Life Sciences Radboudumc 9: Rare cancers RIMLS: Radboud Institute for Molecular Life Sciences |
Organization: | Human Genetics Pathology Internal Medicine |
Journal title: |
Clinical Chemistry
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Volume: | vol. 63 |
Issue: | iss. 2 |
Page start: | p. 503 |
Page end: | p. 512 |
Abstract: |
BACKGROUND: Despite advances in next generation DNA sequencing (NGS), NGS-based single gene tests for diagnostic purposes require improvements in terms of completeness, quality, speed, and cost. Single-molecule molecular inversion probes (smMIPs) are a technology with unrealized potential in the area of clinical genetic testing. In this proof-of-concept study, we selected 2 frequently requested gene tests, those for the breast cancer genes BRCA1 and BRCA2, and developed an automated work flow based on smMIPs. METHODS: The BRCA1 and BRCA2 smMIPs were validated using 166 human genomic DNA samples with known variant status. A generic automated work flow was built to perform smMIP-based enrichment and sequencing for BRCA1, BRCA2, and the checkpoint kinase 2 (CHEK2) c.1100del variant. RESULTS: Pathogenic and benign variants were analyzed in a subset of 152 previously BRCA-genotyped samples, yielding an analytical sensitivity and specificity of 100%. Following automation, blind analysis of 65 in-house samples and 267 Norwegian samples correctly identified all true-positive variants (>3000), with no false positives. Consequent to process optimization, turnaround times were reduced by 60% to currently 10-15 days. Copy number variants were detected with an analytical sensitivity of 100% and an analytical specificity of 88%. CONCLUSIONS: smMIP-based genetic testing enables automated and reliable analysis of the coding sequences of BRCA1 and BRCA2. The use of single-molecule tags, double-tiled targeted enrichment, and capturing and sequencing in duplo, in combination with automated library preparation and data analysis, results in a robust process and reduces routine turnaround times. Furthermore, smMIP-based copy number variation analysis could make independent copy number variation tools like multiplex ligation-dependent probes amplification dispensable.
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