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Reply to: APP gene copy number changes reflect exogenous contamination

Nature volume 584, pages E29–E33 (2020)Cite this article

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The Original Article was published on 19 August 2020

replying to J. Kim et al. Nature https://doi.org/10.1038/s41586-020-2522-3 (2020)

In the accompanying comment1, Kim et al. conclude that somatic gene recombination (SGR) and amyloid precursor protein (APP) genomic complementary DNAs (gencDNAs) in the brain are contamination artefacts and do not naturally exist. We disagree. Here we address the three types of analyses used by Kim et al. to reach their conclusions: informatic contaminant identification, plasmid PCR, and single-cell sequencing. Additionally, Kim et al. requested “reads supporting novel APP insertion breakpoints,” and we now provide ten different examples that support APP gencDNA insertion within eight chromosomes beyond wild-type APP on chromosome 21 from patients with Alzheimer’s disease. If SGR exists, as experimentally supported here and previously2,3, contamination scenarios become moot.

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Fig. 1: Identification of novel APP insertion sites in the human genome.
Fig. 2: Identification of APP gencDNA sequences in ten new whole-exome pull-down datasets from two independent laboratories.
Fig. 3: Five APP gencDNA-supporting reads that span exon–exon junctions and do not contain mouse-specific SNPs.

Data availability

Data from Park et al. were deposited in the National Center for Biotechnology Information Sequence Read Archive database under accession number PRJNA532465. Data from the newly reported full exome pull-down data sets will be provided for the APP locus upon request.

Code availability

The source codes of the customized algorithms are available on GitHub at https://github.com/christine-liu/exonjunction.

References

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Acknowledgements

We thank L. Wolszon and D. Jones for manuscript editing. Research reported in this publication was supported by the NIA of the National Institutes of Health under award numbers R56AG067489 and P50AG005131 (J.C.) and NINDS R01NS103940 (Y.K.). This work was supported by non-federal funds from The Shaffer Family Foundation, The Bruce Ford & Anne Smith Bundy Foundation, and Sanford Burnham Prebys Medical Discovery Institute funds (J.C.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Author notes

  1. These authors contributed equally: Ming-Hsiang Lee, Christine S. Liu

Authors and Affiliations

  1. Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA

    Ming-Hsiang Lee, Christine S. Liu, Yunjiao Zhu, Gwendolyn E. Kaeser, Richard Rivera, William J. Romanow, Yasuyuki Kihara & Jerold Chun

  2. Biomedical Sciences Program, School of Medicine, University of California San Diego, La Jolla, CA, USA

    Christine S. Liu

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Contributions

M.-H.L., Y.K., W.J.R. and R.R. conducted laboratory experiments; C.S.L. and Y.Z. analysed sequencing data; and J.C. conceived and oversaw the experiments. G.E.K, C.S.L, and Y.Z. created figures. All authors wrote and edited the manuscript. This Reply was the work of current laboratory members.

Corresponding author

Correspondence to Jerold Chun.

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Competing interests

Sanford Burnham Prebys Medical Discovery Institute has filed the following patent applications on the subject matter of this publication: (1) PCT application number PCT/US2018/030520 entitled, ‘Methods of diagnosing and treating Alzheimer’s disease’ filed 1 May 2018, which claims priority to US provisional application 62/500,270 filed 2 May 2017; and (2) US provisional application number 62/687,428 entitled, ‘Anti-retroviral therapies and reverse transcriptase inhibitors for treatment of Alzheimer’s disease’ filed 20 June 2018. J.C. is a co-founder of Mosaic Pharmaceuticals.

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Extended data figures and tables

Extended Data Fig. 1 IEJs identified from commercially available long-read transcriptome datasets in two genes other than APP.

Sequences containing IEJs were identified and shown for gene 1 (a) and gene 2 (b). Gene 2 is shown in two parts. Grey dashed lines show ends of RefSeq exons; solid purple lines denote IEJs. All splice isoforms were examined. The Alzheimer brain Iso-Seq dataset was generated by Pacific Biosciences, Menlo Park, CA, and additional information about the sequencing and analysis is available at https://downloads.pacbcloud.com/public/dataset/Alzheimer_IsoSeq_2016/.

Supplementary information

Supplementary Information

This file contains Supplementary Methods; Supplementary Discussion of gencDNAs in blood; Supplementary Discussion of DISH and RISH; and Supplementary References.

Reporting Summary

Supplementary Table 1

Insertion site analysis read alignment information.

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Lee, MH., Liu, C.S., Zhu, Y. et al. Reply to: APP gene copy number changes reflect exogenous contamination. Nature 584, E29–E33 (2020). https://doi.org/10.1038/s41586-020-2523-2

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