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Prevalence of CNV-neutral structural genomic rearrangements in MLH1, MSH2, and PMS2 not detectable in routine NGS diagnostics

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Abstract

Routine diagnostics for colorectal cancer patients suspected of having Lynch-Syndrome (LS) currently uses Next-Generation-Sequencing (NGS) of targeted regions within the DNA mismatch repair (MMR) genes. This analysis can reliably detect nucleotide alterations and copy-number variations (CNVs); however, CNV-neutral rearrangements comprising gene inversions or large intronic insertions remain undetected because their breakpoints are usually not covered. As several founder mutations exist for LS, we established PCR-based screening methods for five known rearrangements in MLH1, MSH2, or PMS2, and investigated their prevalence in 98 German patients with suspicion of LS without a causative germline variant or CNV detectable in the four MMR genes. We found no recurrence of CNV-neutral structural rearrangements previously described: Neither for two inversions in MLH1 (exon 1 and exon 16–19) within 33 MLH1-deficient patients, nor for two inversions in MSH2 (exon 1–7 and exon 2–6) within 48 MSH2-deficient patients. The PMS2 insertion in intron 7 was detected in one of 17 PMS2-deficient patients. None of the four genomic inversions constitutes a founder event within the German population, but we advise to test the rare cases with unsolved PMS2-deficiency upon the known insertion. As a next diagnostic step, tumour tissue of the unsolved patients should be sequenced for somatic variants, and germline analysis of additional genes with an overlapping clinical phenotype should be considered. Alternatively, full-length cDNA analyses may detect concealed MMR-defects in cases with family history.

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Abbreviations

CNV(s):

Copy-number variation(s)

CRC:

Colorectal cancer

LS:

Lynch-syndrome

MLPA:

Multiplex ligation-dependent probe amplification

MMR:

DNA mismatch repair

NGS:

Next-generation-sequencing

PCR:

Polymerase chain reaction

SNV:

Single nucleotide variant

SVA:

SINE–VNTR–Alu

VUS:

Variant of uncertain significance

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Acknowledgements

We thank the Deutsche Krebshilfe e.V. and the Wilhelm Sander-Stiftung for funding this work. We also thank the medical doctors and patients for their participation in this study, as well as the ERN GENTURIS and the German Consortium for Familial Intestinal Cancer for their support. Furthermore, we appreciate Professor Robyn L. Ward and Dr. Mathew A. Sloane from the University of New South Wales, Sydney, Australia and Dr. Jennifer Rhees from the Gastrointestinal Cancer Research Laboratory, Baylor University Medical Center, Dallas, TX, USA for generously providing us a positive control DNA sample for a MSH2 inversion each.

Funding

This study was funded by the Deutsche Krebshilfe e.V. grant number 80935055 and the Wilhelm Sander-Stiftung Grant Number 80735127.

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Authors and Affiliations

  1. Medizinische Klinik Und Poliklinik IV, Campus Innenstadt, Klinikum Der Universität München, Ziemssenstr. 1, 80336, Munich, Germany

    Monika Morak, Verena Steinke-Lange, Trisari Massdorf, Anna Benet-Pages, Melanie Locher, Andreas Laner & Elke Holinski-Feder

  2. MGZ – Medizinisch Genetisches Zentrum, Bayerstr. 3-5, 80335, Munich, Germany

    Monika Morak, Verena Steinke-Lange, Trisari Massdorf & Elke Holinski-Feder

  3. Institute of Human Genetics, University of Bonn, Bonn, Germany

    Katrin Kayser & Stefan Aretz

  4. Center for Hereditary Tumour Syndromes, University Hospital Bonn, Bonn, Germany

    Stefan Aretz

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  1. Monika Morak
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  2. Verena Steinke-Lange
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Correspondence to Monika Morak or Elke Holinski-Feder.

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10689_2020_159_MOESM1_ESM.tif

Schematic presentation of the PCR-based detection method used as a screening for rearrangements, with one example each for an inversion (a) and an insertion (b). In the wildtype allele above, the primers of the control PCR are depicted as arrows in green and yellow. Here, the breakpoint(s) of the inversion or insertion are marked with red flashes within the wildtype allele, and the allele with the rearrangement is shown below. For the newly generated breakpoints, rearrangement-specific PCR amplifications were set up. (a) The inversion-specific fusion breakpoints are detected by PCR amplifications, one using the green primer pair, and the other using the yellow primer pair in the rearrangement allele. (b) The insertion of the 2.2 kb SVA element in PMS2 intron 7 is detected by amplification of an insertion-specific PCR product using primer combination in green and red. (TIF 831 kb)

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Morak, M., Steinke-Lange, V., Massdorf, T. et al. Prevalence of CNV-neutral structural genomic rearrangements in MLH1, MSH2, and PMS2 not detectable in routine NGS diagnostics. Familial Cancer 19, 161–167 (2020). https://doi.org/10.1007/s10689-020-00159-4

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