GDF15 and ACE2 stratify COVID19 patients according to severity while ACE2 mutations increase infection susceptibility

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1. 

   ScreenIT

   May 9, 2022

   SciScore for 10.1101/2022.05.06.490907: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	IRB: Ethical approval for this project (IB 4165/20 PI, 6 April 2020) was obtained from the ethics committee of the Balearic Islands and waived the requirement for informed consent, due to the emergency situation. Consent: Ethical approval for this project (IB 4165/20 PI, 6 April 2020) was obtained from the ethics committee of the Balearic Islands and waived the requirement for informed consent, due to the emergency situation.
   Sex as a biological variable	not detected.
   Randomization	The reaction mixture contained 250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2, 2.5 µM random hexamers, 500 µM of each dNTP, 20 U RNase inhibitor, 10 mM DTT, and 200 U M-Mlv reverse transcriptase.
   Blinding	not detected.

   SciScore for 10.1101/2022.05.06.490907: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	IRB: Ethical approval for this project (IB 4165/20 PI, 6 April 2020) was obtained from the ethics committee of the Balearic Islands and waived the requirement for informed consent, due to the emergency situation. Consent: Ethical approval for this project (IB 4165/20 PI, 6 April 2020) was obtained from the ethics committee of the Balearic Islands and waived the requirement for informed consent, due to the emergency situation.
   Sex as a biological variable	not detected.
   Randomization	The reaction mixture contained 250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2, 2.5 µM random hexamers, 500 µM of each dNTP, 20 U RNase inhibitor, 10 mM DTT, and 200 U M-Mlv reverse transcriptase.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   To generate SARS-Cov-2 pseudovirus, Human embryonic kidney 293 (HEK 293, ATCC® CRL-1573™) cells were seeded in a 10 cm2-plate at 4.5×106 cells/plate, in DMEM (ThermoFisher Scientific, #11995073	HEK 293 suggested: ATCC Cat# CRL-1573, RRID:CVCL_0045)
   Pseudovirus entry assay: A549 cells (ATCC® CCL-185) seeded in 6-multiwell plates at 0.23 × 106 cells/well in DMEM (10% FBS and 1x Normocin), and grown overnight at 37 °C, 85%	A549 suggested: None
   Recombinant DNA
   Sentences	Resources
   psPAX2 was a gift from Dr Didier Trono	psPAX2 suggested: RRID:Addgene_12260)
   Addgene plasmid # 12260); pLV-mCherry was a gift from Dr Pantelis Tsoulfas	pLV-mCherry suggested: RRID:Addgene_36084)
   Addgene plasmid # 36084); pcDNA3 mCherry was a gift from Dr Scott Gradia	pcDNA3 suggested: RRID:Addgene_15475)
   (Addgene plasmid # 30125); pcDNA3.1(+)eGFP was a gift from Dr Jeremy Wilusz	pcDNA3.1 suggested: RRID:Addgene_79663)
   Addgene plasmid # 129020); pcDNA3.1-SARS2-Spike was a gift from Dr Fang Li	pcDNA3.1-SARS2-Spike suggested: RRID:Addgene_145032)
   (Addgene plasmid # 145032); pcDNA3.1-ACE2-GFP was a gift from Dr Utpal Pajvani	pcDNA3.1-ACE2-GFP suggested: RRID:Addgene_154962)
   Next day, HEK 293 cells were then cotransfected with 15 μg of psPAX2 : pcDNA3.1SpikeVOCs : pLV-mCherry at ratio 1:1:1 (i.e. 5:5:5 μg) using Lipofectamin™ 3000 transfection reagent (Thermo-Fisher Scientific, #L3000015)	pcDNA3.1SpikeVOCs suggested: None
   Next day, cells were transfected with either pcDNA3.1(+)eGFP (control) or pcDNA3.1-GFP-ACE2 expressing the ACE2 SNPs.	pcDNA3.1-GFP-ACE2 suggested: None
   Software and Algorithms
   Sentences	Resources
   100k Genomes Project (100KGP) as the main sources of aggregated data for ACE2 protein altering variations in populations groups across the world.	100k Genomes Project suggested: (MedBlast, RRID:SCR_008202)
   PCR product was purified by clean-up (FAGCK001, Favorgen) and further sequenced by Macrogen.	Macrogen suggested: (Macrogen, RRID:SCR_014454)
   Statistical analyses were performed using R Studio version 3.5.2 of the R programming language (R Project for Statistical Computing; R Foundation, Vienna, Austria). and Stata v17.0 program.	R Project for Statistical suggested: (R Project for Statistical Computing, RRID:SCR_001905)

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   Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   Our study has limitations. First the study population is small, including patients from Hospital Son Llatzer and Hospital Son Espases, two of the main hospitals in the Balearic Health System. There might be a selection bias when identifying factors that differ between patients with COVID-19, although COVID-19 patients were matched for age and sex as well with the control group. Second, it is a retrospective study conducted in an emergency situation, and in which not all the clinical characteristics of the patients were recorded. Third, some patients had elevated biomarker data in some laboratory measurements, as well as in GDF15 and ACE2 levels, and we did not exclude them from the analysis due to small sample size and because those numbers were physiological and not due to technical errors. Finally, the study is cross-sectional and no causal inferences can be made. Despite the limitations, our study provides convincing evidence that in patients with COVID-19, the levels of GDF15 and ACE2 could be associated with increased inflammation and disease severity while ACE2 missense SNPs might be linked to infection susceptibility. In conclusion, critically ill patients with COVID-19 present higher levels of GDF15 and ACE2, as well as acute inflammation. These two proteins might be of importance because of its association with disease severity in patients infected with SARS-CoV-2. Our results suggest that certain genetic variations in ACE2 might modulate susceptibility to SARS-CoV-2...

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   Results from rtransparent:

   • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
   • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
   • No protocol registration statement was detected.

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   Results from scite Reference Check: We found no unreliable references.

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2. 

   Version published to 10.1101/2022.05.06.490907v1 on bioRxiv

   May 6, 2022