The gene for the amyloid precursor protein (APP) shows somatic gene recombination in neurons, and the abundance and diversity of APP variants is increased in neurons from individuals with Alzheimer’s disease.
Mutations that occur in cells of the body, called somatic mutations, cause human diseases including cancer and some neurological disorders. In a recent study published in Nature, Lee et al. (hereafter “the Lee study”) reported somatic copy number gains of the APP gene, a known risk locus of Alzheimer’s disease (AD), in the neurons of AD-patients and controls (69% vs 25% of neurons with at least one APP copy gain on average). The authors argue that the mechanism of these copy number gains was somatic integration of APP mRNA into the genome, creating what they called genomic cDNA (gencDNA). We reanalyzed the data from the Lee study, revealing evidence that APP gencDNA originates mainly from contamination by exogenous APP recombinant vectors, rather from true somatic retrotransposition of endogenous APP . Our reanalysis of two recent whole exome sequencing (WES) datasets—one by the authors of the Lee study and the other by Park et al.—revealed that reads claimed to support APP gencDNA in AD samples resulted from contamination by PCR products and mRNA, respectively. Lastly, we present our own single-cell whole genome sequencing (scWGS) data that show no evidence for somatic APP retrotransposition in AD neurons or in neurons from normal individuals of various ages.
In the accompanying comment, Kim et al . concluded that somatic gene recombination ( SGR ) and amyloid precursor protein ( APP ) genomic complementary DNAs ( gencDNAs ) in brain are contamination artifacts 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 10 different examples that support APP gencDNA insertion within eight chromosomes beyond wildtype APP on chromosome 21 from Alzheimer’s disease ( AD ) samples. If SGR exists as experimentally supported here and previously,, contamination scenarios become moot. Our informatic analyses of data generated by an independent laboratory (Park et al.) , complement and are entirely consistent with what Lee et al . presented via nine distinct lines of evidence, in addition to three from a prior publication. Plasmid contamination was identified in a single pull-down dataset after publication of Lee et al .; however subsequent analyses did not alter any of our conclusions including those of our prior publications, and plasmid contamination-free replication of this approach by ourselves and others supported the original conclusions. Novel retro-insertion sites, alterations of APP gencDNA number and form with cell type from the same brain and pathogenic SNVs occurring only in AD, all support the existence of APP gencDNAs produced by SGR.
### Competing Interest Statement
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. JC is a co-founder of Mosaic Pharmaceuticals.
Evidence that APP gene copy number changes reflect recombinant vector contamination
Junho Kim, Boxun Zhao, August Yue Huang, Michael B. Miller, Michael A. Lodato, Christopher A. Walsh, Eunjung Alice Lee
bioRxiv 706788; doi: https://doi.org/10.1101/706788
Reply: Evidence that APP gene copy number changes reflect recombinant vector contamination
Ming-Hsiang Lee, Christine S. Liu, Yunjiao Zhu, Gwendolyn E. Kaeser, Richard Rivera, William J. Romanow, Yasuyuki Kihara, Jerold Chun
bioRxiv 730291; doi: https://doi.org/10.1101/730291
ALZFORUM News, Rogue APP Claim Embroiled in Contamination Concerns, https://www.alzforum.org/news/research-news/rogue-app-claim-embroiled-contamination-concerns