Presymptomatic Increase of an Extracellular RNA in Blood Plasma Associates with the Development of Alzheimer‚Äôs Disease
Authors: Zhangming Yan
Edward H Koo
Sheng Zhong
Qiuyang Wu
Zhen Bouman Chen
Zixu Zhou
Journal: Current Biology (2020)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Alzheimer‚Äôs disease; PHGDH; SILVER-seq; biomarker; brain
Affiliations: Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA., Genemo Inc., 3210 Merryfield Row, San Diego, CA 92121, USA., Beckman Research Institute, City of Hope, Duarte, CA 91010, USA., Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093, USA., Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA; Genemo Inc., 3210 Merryfield Row, San Diego, CA 92121, USA. Electronic address: .

Abstract:  The extracellular RNAs (exRNAs) from human biofluid have recently been systematically characterized. However, the correlations of biofluid exRNA levels and human diseases remain largely untested. Here, considering the unmet need for presymptomatic biomarkers of sporadic Alzheimer's disease (AD), we leveraged the recently developed SILVER-seq (small-input liquid volume extracellular RNA sequencing) technology to generate exRNA profiles from a longitudinal collection of human plasma samples. These 164 plasma samples were collected from research subjects 70 years or older with up to 15 years of clinical follow-up prior to death and whose clinical diagnoses were confirmed by pathological analysis of their post mortem brains. The exRNAs of AD-activated genes and transposons in the brain exhibited a concordant trend of increase in AD plasma in comparison with age-matched control plasma. However, when we required statistical significance with multiple testing adjustments, phosphoglycerate dehydrogenase (PHGDH) was the only gene that exhibited consistent upregulation in AD brain transcriptomes from 3 independent cohorts and an increase in AD plasma as compared to controls. We validated PHGDH's serum exRNA and brain protein expression increases in AD by using 5 additional published cohorts. Finally, we compared the time-course exRNA trajectories between "converters" and controls. Plasma PHGDH exRNA exhibited presymptomatic increases in each of the 11 converters during their transitions from normal to cognitive impairment but remained stable over the entire follow-up period in 8 out of the 9 control elderly subjects. These data suggest the potential utilities of plasma exRNA levels for screening and longitudinal exRNA changes as a presymptomatic indication of sporadic AD. 

Retracted: No

PubPeer comments:
 Yan et al. introduced their library preparation method, called SILVER-Seq, in a paper named *Extracellular RNA in a single droplet of human serum reflects physiologic and disease states* (Zhou et al., 2019), published in PNAS in September 2019. Since then, two Letters to the Editor have been written in response: one reporting a possible **batch effect** (Hartl and Gao, 2020), another one pointing at excessive **DNA contamination** (Verwilt et al., 2020). The authors replied to both Letters (Zhou et al., 2020a, 2020b). In order to evaluate if the new data generated in this paper are also plagued by DNA contamination, we ran the same pipeline using the online available data. Again, we noted a very low average fraction of exonic (5.13%) and splice-junction reads (0.41%), similar to our earlier findings (Verwilt et al., 2020). **These findings very likely suggest that the data generated in this paper mainly originate from DNA instead of cell-free RNA**. Therefore, it would have been ideal if these quality control measures were included or at least mentioned in this manuscript of Yan et al.  **Figure 1:** Fraction of reads mapped against exonic, intronic and intergenic regions. The median values are printed.  **Figure 2:** Fraction of splice and nonsplice reads. The median values are printed. Hartl, C., and Gao, Y. (2020). Clarifying the effect of library batch on extracellular RNA sequencing. Proc. Natl. Acad. Sci. U. S. A. 117, 1849u20131850. Verwilt, J., Trypsteen, W., Van Paemel, R., De Preter, K., Giraldez, M.D., Mestdagh, P., and Vandesompele, J. (2020). When DNA gets in the way: A cautionary note for DNA contamination in extracellular RNA-seq studies. Proc. Natl. Acad. Sci. 117, 18934u201318936. Zhou, Z., Wu, Q., Yan, Z., Zheng, H., Chen, C.-J., Liu, Y., Qi, Z., Calandrelli, R., Chen, Z., Chien, S., et al. (2019). Extracellular RNA in a single droplet of human serum reflects physiologic and disease states. Proc. Natl. Acad. Sci. 116, 19200u201319208. Zhou, Z., Wu, Q., Yan, Z., Zheng, H., Chen, C.J., Liu, Y., Qi, Z., Calandrelli, R., Chen, Z., Chien, S., et al. (2020a). Lack of between-batch difference in the distributions of measured extracellular RNA levels. Proc. Natl. Acad. Sci. U. S. A. 117, 1851u20131852. Zhou, Z., Wu, Q., Yan, Z., Zheng, H., Chen, C.J., Liu, Y., Qi, Z., Calandrelli, R., Chen, Z., Chien, S., et al. (2020b). Reply to Verwilt et al.: Experimental evidence against DNA contamination in SILVER-seq. Proc. Natl. Acad. Sci. U. S. A. 117, 18937u201318938. , 
 #1 Dear Dr. Verwilt, Thank you for your comment. We have provided u201cExperimental evidence against DNA contamination in SILVER-sequ201d in the following open access paper: https: www.pnas.org content pnas 117 32 18937.full.pdf. These experimental data failed to support the two metrics you mentioned as good quality control metrics in the context of our applications. We feel it is immature to include these two metrics in our paper before these metrics are fully validated, potentially by future work in clarifying the human exRNA biogenesis and retention mechanisms. ,