1. Genetics and Genomics
  2. Medicine
Download icon

Patient-specific genomics and cross-species functional analysis implicate LRP2 in hypoplastic left heart syndrome

  1. Jeanne L Theis
  2. Georg Vogler
  3. Maria A Missinato
  4. Xing Li
  5. Tanja Nielsen
  6. Xin-Xin I Zeng
  7. Almudena Martinez-Fernandez
  8. Stanley M Walls
  9. Anaïs Kervadec
  10. James N Kezos
  11. Katja Birker
  12. Jared M Evans
  13. Megan M O'Byrne
  14. Zachary C Fogarty
  15. André Terzic
  16. Paul Grossfeld
  17. Karen Ocorr
  18. Timothy J Nelson  Is a corresponding author
  19. Timothy M Olson  Is a corresponding author
  20. Alexandre R Colas
  21. Rolf Bodmer  Is a corresponding author
  1. Cardiovascular Genetics Research Laboratory, United States
  2. Development, Aging and Regeneration, Sanford Burnham Prebys Medical Discovery Institute, United States
  3. Division of Biomedical Statistics and Informatics, Mayo Clinic, United States
  4. Department of Cardiovascular Medicine, Mayo Clinic, United States
  5. Department of Molecular and Pharmacology and Experimental Therapeutics, Mayo Clinic, United States
  6. Center for Regenerative Medicine, Mayo Clinic, United States
  7. Division of Pediatric Cardiology, Department of Pediatric and Adolescent Medicine, Mayo Clinic, United States
  8. University of California San Diego, Rady’s Hospital, United States
  9. Division of General Internal Medicine, Mayo Clinic, United States
Tools and Resources
Cite this article as: eLife 2020;9:e59554 doi: 10.7554/eLife.59554
4 figures, 3 videos, 2 tables and 10 additional files

Figures

Figure 1 with 2 supplements
Family-based iPSC characterization for HLHS.

(A) Pedigree of family 5H: proband with HLHS (black symbol), relatives without CHD (white symbols), miscarriages (gray diamonds). (B) Schematic for family-based iPSC production and characterization. (C) Whole-genome RNA sequencing identified 1401 concordantly DETs between proband and parents. (D) KEGG pathway analysis shows enrichment of DETs in TP53 pathway. (E) Heatmap of p53 signaling pathway-associated genes in probands vs parents. (F) Schematic describing EdU-incorporation assay in hiPSC-CMs. 5000 cells/well were plated in 384 well plates. After 48 hr EdU was added to the media and left incorporate for 24 hr. Cells were then fixed and stained (G) Graph representing quantification of EdU+ cardiomyocytes in HLHS 5H family-derived iPSC-CMs. ***p<0.001 one-way ANOVA. (H) Representative images of iPSC-CMs derived from mother (Top) and proband (Bottom), stained for EdU, ACTN1 and DAPI. Scale bar: 50 µm. (I) Quantification of EdU-incorporation assay in 5H proband iPSC-CM upon KD of TP53 or CDKN1A. ****p<0.0001, one-way ANOVA. (J) Representative images of 5H proband iPSC-CM stained for EdU and ACTN1 upon KD of TP53 or CDKN1A at day 28. Scale bar: 50 µm.

Figure 1—figure supplement 1
Cell cycle activity is altered in HLHS patient-derived iPSCs and CMs.

(A) Heatmap of negative regulation of cell proliferation-associated genes from RNA-seq experiments in proband vs parents. (B) Heatmap of cell cycle arrest-associated genes. (C) Heatmap of positive regulation of apoptosis-associated genes.

Figure 1—figure supplement 2
Cell cycle activity is altered in HLHS patient-derived iPSCs and CMs.

(A) Quantification of percentage of EdU+ cells in 75H and 151H iPSCs. Proband iPSCs show reduced EdU incorporation compared to the parents. **p<0.01, ****p<0.0001 in 75H family. ##p<0.01 in the151H family. One-way ANOVA. (B) Representative images of 75H family iPSCs labeled for EdU and DAPI. Of note, the yield of patient-derived iPSC was relatively low. Scale bar: 50 µm.

Figure 2 with 4 supplements
Whole-genome and RNA sequencing identify HLHS candidate genes.

(A) An iterative, family-based variant filtering approach based on rarity, functional impact, and mode of inheritance and RNA sequencing data were used to filter for transcriptional differences yielding 10 candidate genes. Candidate genes were further tested in hiPSC-CM and in vivo model. (B) Human candidate genes and corresponding Drosophila ortholog as determined by DIOPT score (*confidence score: number of databases reporting orthology). Listed are heart phenotypes upon gene candidate KD. (C,D) Example of fly hearts heterozygous for LRP2/mgl show increased end-diastolic diameters (EDD, measured at green line in D). Wilcoxon rank sum test: ***p<0.001. (E) Graph representing EdU-incorporation assay results of candidate gene KD in hiPSC-CM. KD of APOB (red bar) or LRP2 (green bar) reduced EdU incorporation. **p<0.01 one-way ANOVA. (F) Representative images of hiPSC-CMs stained for EdU, ACTN1 and DAPI. Scale bar: 50 µm. (G) qPCR results of TP53 and CDKN1A in hiPSC-CM upon KD of APOB or LRP2. *p<0.05 one-way ANOVA.

Figure 2—figure supplement 1
Identification of HLHS candidate genes from whole-genome and RNA sequencing.

An iterative, family-based variant filtering approach based on rarity, functional impact, and mode of inheritance yielded 61 candidate genes. RNA sequencing data from d0 iPSC and d25 differentiated iPSC were used to filter for transcriptional differences yielding ten candidate genes.

Figure 2—figure supplement 2
Cell cycle activity is altered in HLHS patient-derived iPSCs and CMs.

qPCR of the 10 candidate genes in the ‘5H-family’ iPSC-CMs at day 25 of differentiation. Proband cells show downregulation of all 10 candidate genes compared to the mother’s cells (*) and 7/10 compared to the father’s cells (#). Data are normalized to the cells derived from the mother. ***p<0.001; ****p<0.0001; ##p<0.01; ####p<0.0001; n.s.: not significant. One-way ANOVA.

Figure 2—figure supplement 3
Phenotypic assessment of HLHS candidate genes in Drosophila adult hearts.

(A) Schematic of the Drosophila adult heart assay. (B) Human candidate genes and corresponding Drosophila ortholog as determined by DIOPT score (*confidence score: number of databases reporting orthology). Listed are heart phenotypes upon knockdown (KD) in wild-type or NKX2-5/tin+/-heterozygous background. (C–E) RNAi-induced arrhythmicity and M-modes observed with LRP2/mgl and APOB/apolpp KD. (F–H) Heart size (EDD: end-diastolic diameter) alterations upon RNAi-KD of JPT1, HSPG2 and SDHD (also in NKX2-5/tin heterozygous background). Wilcoxon rank sum test: *p<0.05, **p<0.01, **p<0.001.

Figure 2—figure supplement 4
LRP2 and APOB KD reduces total nuclei and affect cell cycle in hiPSC-CMs.

(A) qPCR showing KD efficiency for the 10 candidate genes in day 28 hiPSC-CM. For all the 10 genes tested, siRNAs KD efficiency is about 50%. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, Student’s t-test. (B) Quantification of total nuclei in day 28 hiPSC-CMs transfected with siRNAs directed to ten candidate HLHS genes. LRP2 and APOB KD reduce total cells number. (C) qPCR of tumor suppressors/cell cycle inhibitor genes in day 28 hiPSC-CMs upon KD of LRP2 or APOB. Tumor suppressors/cell cycle inhibitors are upregulated upon LRP2/APOB KD compared to control. *p<0.05, Student’s t-test. (D) qPCR of cell cycle genes in day 28 hiPSC-CMs upon KD of APOB or LRP2. Cell cycle genes are downregulated upon LRP2/APOB KD. *p<0.05, Student’s t-test.

Figure 3 with 1 supplement
Identification of LRP2 as potential HLHS candidate gene.

(A) Cohort-wide analysis of LRP2 variants shows significant enrichment for SNVs in HLHS patients compared to control populations. Variants (blue/magenta) are found throughout LRP2 protein. (B) Table listing the HLHS families carrying LRP2 variants. (C,D) qPCR of LRP2 in 5H family (C) and in 49H family (D) showing LRP2 downregulation in carrier parent and proband compared to the non-carrier parent. ****p<0.0001 one-way ANOVA #p<0.05 one-way ANOVA. (E) Cardiomyocyte count in zebrafish morphants at 72 hpf were significantly reduced in the ventricle. (F) Atrial cardiomyocyte number was also reduced in morphants but to a lesser extent than in ventricles. *p<0.05; ****p<0.0001 unpaired two-tail Student t-test. (G, top panel) embryonic fish hearts were visualized by EGFP expression in the myl7:EGFP transgenic background (green) at 72 hpf. lrp2a morphant hearts were dysmorphic and much smaller (arrow) compared to controls. (G, lower panel) myl7:H2A-mCherry transgenic background identifies cardiomyocyte nuclei used for quantifying cardiomyocytes during development in E and F. Dotted traces outline the ventricles in G. Scale bars: 30 μm.

Figure 3—figure supplement 1
lrp2a KD and CRISPR causes reduced contractility and bradycardia in zebrafish larva.

(A) Uninjected zebrafish larva, (B) lrp2a morpholinos (MO) (1 pl of 2 ng/µl) injected larva and (C) lrp2a CRISPR genome edited F0 larva all exhibit relatively normal body morphology at 72 hpf, note the pericardial edema evident in morphant and F0 mutant (arrows). Scale bar: 200 µm. (D) End-diastolic surface area and (E) End Systolic surface area in atria and ventricles determined from high-speed movies of beating hearts at 72 hpf. (F) Contractility, measured as fractional area change, was significantly reduced in ventricles from both morphants and mutants. (G) Heart period was significantly lengthened in morphants and mutant larvae. (D–G) Two doses of lrp2a MO were injected (1 pl of 1 ng/µl or 2 ng/µl). For CRISPR/Cas9 and guide RNA concentration, please see detailed information in the Materials and Methods section. One-way ANOVA, Dunnett’s multiple comparisons post hoc test.

Potential role for SHH, WNT and LRP2 in HLHS.

(A) A gene network integrating family-centric HLHS candidate genes with heart development. ORANGE – genes with cardiac phenotypes in iPSC/Drosophila assays. YELLOW – other candidate genes with Drosophila phenotypes. RED – Genes up-regulated in proband iPSC-CMs vs. parents. BLUE – Genes downregulated downregulated in proband iPSCs vs. parents. (B,C) qPCR for FZD10, a WNT-pathway-associated gene, (B) and for PTCH1, a SHH pathway-associated gene (C) upon LRP2 KD in hiPSC-CM. **p<0.01, *p<0.05 Student’s t-test. (D) Quantification of EdU- incorporation assay in hiPSC-CM upon LRP2 KD in combination with BIO, a WNT inhibitor. ***or ###p<0.001, ****p<0.0001, one-way ANOVA. (E) Representative images of hiPSC-CM stained for EdU and ACTN1. Scale bars: 50 µm. (F) Quantification of EdU-incorporation assay in hiPSC-CM upon LRP2 KD in combination with PTCH1 KD, a SHH-associated gene. ***p<0.001, ****p<0.0001, one-way ANOVA. (G) Representative images of hiPSC-CM stained for EdU and ACTN1. Scale bars: 50 µm.

Videos

Video 1
Dissected adult fly heart showing rhythmic beating pattern.

Representative heart movies of dissected adult females showing arrhythmic beating pattern in APOB-RNAi (Video 2) and LRP2-RNAi (Video 3) compared to control hearts (Video 1). All movies are imaged at 140 frames/sec.

Video 2
APOB-RNAi causes arrhythmia in dissected adult fly hearts.
Video 3
LRP2-RNAi causes arrhythmia in dissected adult fly hearts.

Tables

Table 1
Recessive Variants Identified in 10 Candidate Genes.
GeneMode of inheritanceFunctional impactTranscript variantProtein variantInheritanceGenotype in brother (II.1)Genotype in sister (II.2)gnomAD* MAF (%)dbSNP ID
HSPG2Cmpd Hetmissensec.2074G > A; c.2077G > Ap.V692M; p.V693MMaternalWTWT0.288143669458
missensec.326G > Ap.R109QPaternalHetHet0773796176
promoterc.-227C > APaternalWTWT1.392566166086
SLC9A1Cmpd Hetpromoterc.-906T > CPaternalHetHet1.227114101904
promoterc.-947T > GMaternalWTWT27.17511588974
promoterc.-1085A > GPaternalHetHet0.841116299278
ENCODE TFBSc.-1138C > TPaternalHetHet0.9375089536
promoterc.-1311G > APaternalHetHet0.9377414471
APOBCmpd Hetmissensec.13441G > Ap.A4481TMaternalWTHet2.4751801695
missensec.751G > Ap.A251TPaternalHetWT0.07161741625
LRP2Cmpd Hetmissensec.9613A > Gp.N3205DMaternalWTWT0.40735734447
missensec.170C > Tp.A57VPaternalWTWT0.032115350461
SDHDCmpd Hetpromoterc.-815G > C; c.129+547C > GMaternalHetWT0.573117661257
ENCODE TFBSc.-205G > A; c.66C > Tp.A22APaternalWTWT0.24161734353
missensec.34G > A; c.-173C > Tp.G12SMaternalHetWT0.72934677591
PRTGCmpd HetmicroRNA Binding Sitec.*3501T > GPaternalHetWT0.73977181316
microRNA Binding Sitec.*2678A > GMaternalWTHet0.019756136447
HN1Cmpd HetENCODE TFBSc.56+617C > T; c.-903C > T; c.-178+617C > T; c.-590C > TMaternalHetWT3.764117213586
promoterc.-1748A > C; c.-719A > C; c.-486A > CPaternalWTHet0.81673995795
SIK1Hom Recmissensec.2087C > Tp.P696LMaternal and PaternalWTHet1256991707
ELF4X-Linkedmissensec.1144G > Ap.V382IMaternalWTHet0.025148953158
HS6ST2X-Linkedmissensec.948–40041G > A; c.1046G > Ap.R349QMaternalWTHet0.146201239951
  1. Cmpd Het, compound heterozygous; Het, heterozygous; Hom Rec, homozygous recessive; MAF, minor allele frequency; WT, wild- type.

    *At study initiation the ESP database was used to set the 3% allele frequency filter. Updated frequencies are shown based on the newer gnomAD database curation which would now eliminate SLC9A1 and HN1 as candidate genes.

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Genetic reagent (D. melanogaster)Hand4.2-Gal4NAPMID:16467358NA
Genetic reagent (D. melanogaster)UAS-trolRNAiVienna Drosophila Resource Center (VDRC)FBst0454629v22642
Genetic reagent (D. melanogaster)UAS-CG1943RNAiVienna Drosophila Resource Center (VDRC)FBst0453803v20758
Genetic reagent (D. melanogaster)UAS-apolppRNAiVienna Drosophila Resource Center (VDRC)FBst0470481v6878
Genetic reagent (D. melanogaster)UAS-Hs6stRNAiVienna Drosophila Resource Center (VDRC)FBst0464695v42658
Genetic reagent (D. melanogaster)UAS-mglRNAiVienna Drosophila Resource Center (VDRC)FBst0461660v36389
Genetic reagent (D. melanogaster)UAS-SdhdRNAiVienna Drosophila Resource Center (VDRC)FBst0456581v26776
Genetic reagent (D. melanogaster)UAS-Nhe2RNAiVienna Drosophila Resource Center (VDRC)FBst0477879v106053
Genetic reagent (D. melanogaster)UAS-JupiterRNAiVienna Drosophila Resource Center (VDRC)FBst0455704v25044
Genetic reagent (D. melanogaster)UAS-Eip74EFRNAiVienna Drosophila Resource Center (VDRC)FBst0477129v105301
Genetic reagent (D. melanogaster)UAS-Sik2RNAiVienna Drosophila Resource Center (VDRC)FBst0456442v26496
Genetic reagent (D. melanogaster)mglMI14318Bloomington Drosophila Stock Center (BDSC)FBal0302551BL-59689
Genetic reagent (D. melanogaster)tin346NAFBal0035787NA
Strain, strain background (D. rerio)Oregon AB wild-typeA commonly used wild-type strain
Strain, strain background (D. rerio)Tg(myl7:EGFP)twu277Tsai Lab, National Taiwan UniversityPMID:12950077A transgenic line of zebrafish labeled with heart-specific EGFP fluorescence.
Strain, strain background (D. rerio)Tg(myl7:H2A-mCherry)sd12Yelon Lab, University of California, San DiegoPMID:24075907A transgenic line of zebrafish specifically expressing mCherry in cardiomyocyte nuclei
Antibodymouse monoclonal anti-ACTN1SigmaA78111:800
Antibodydonkey polyclonal anti-mouse Alexa fluor 568InvitrogenA100371:1000
Antibodychicken polyclonal anti-GFPAves LabsGFP-10201:300
Antibodyrabbit polyclonal abit-mCherryRockland600–401 P16S1:200
Antibodydonkey polyclonal anti-chicken AlexaFluor 488Jackson ImmunoResearch703-545-1551:200
Antibodydonkey polyclonal anti-rabbit AlexaFluor 568InvitrogenA100421:200
OtherDAPI (iPSC) 500 mg/mLSigmaD95421:1000
OtherDAPI (Zebrafish) 500 mg/mLInvitrogenD13061:200
Sequence-based reagentLRP2 siRNAEntrez Gene ID: 4036DharmaconOn-Target plus, SmartPool
Sequence-based reagentAPOB siRNAEntrez Gene ID: 338DharmaconOn-Target plus, SmartPool
Sequence-based reagentPTCH1 siRNAEntrez Gene ID: 5727DharmaconOn-Target plus, SmartPool
Sequence-based reagentTP53 siRNAEntrez Gene ID: 7157DharmaconOn-Target plus, SmartPool
Sequence-based reagentCDKN1A siRNAEntrez Gene ID: 1026DharmaconOn-Target plus, SmartPool
Sequence-based reagentELF4 siRNAEntrez Gene ID: 2000DharmaconOn-Target plus, SmartPool
Sequence-based reagentJPT1 siRNAEntrez Gene ID: 51155DharmaconOn-Target plus, SmartPool
Sequence-based reagentHS6ST2 siRNAEntrez Gene ID: 90161DharmaconOn-Target plus, SmartPool
Sequence-based reagentHSPG2 siRNAEntrez Gene ID: 3339DharmaconOn-Target plus, SmartPool
Sequence-based reagentPRTG siRNAEntrez Gene ID: 283659DharmaconOn-Target plus, SmartPool
Sequence-based reagentSDHD siRNAEntrez Gene ID: 6392DharmaconOn-Target plus, SmartPool
Sequence-based reagentSIK1 siRNAEntrez Gene ID: 150094DharmaconOn-Target plus, SmartPool
Sequence-based reagentSLC9A1 siRNAEntrez Gene ID: 6548DharmaconOn-Target plus, SmartPool
Sequence-based reagentCDHHs00170423_m1IDT Integrated DNA technologies, Coralville, IAcharacterization of the pluripotent state
Sequence-based reagentDNMT3Hs01003405_m1IDT Integrated DNA technologies, Coralville, IAcharacterization of the pluripotent state
Sequence-based reagentDPPA2Hs00414521_g1IDT Integrated DNA technologies, Coralville, IAcharacterization of the pluripotent state
Sequence-based reagentDPPA5Hs00988349_g1IDT Integrated DNA technologies, Coralville, IAcharacterization of the pluripotent state
Sequence-based reagentERASHs.PT.45.4849266.gIDT Integrated DNA technologies, Coralville, IAcharacterization of the pluripotent state
Sequence-based reagentGDF3Hs00220998_m1IDT Integrated DNA technologies, Coralville, IAcharacterization of the pluripotent state
Sequence-based reagentOCT-4Hs.PT.45.14904310.gIDT Integrated DNA technologies, Coralville, IAcharacterization of the pluripotent state
Sequence-based reagentREXO1Hs.PT.45.923095.gIDT Integrated DNA technologies, Coralville, IAcharacterization of the pluripotent state
Sequence-based reagentSALL4Hs00360675_m1IDT Integrated DNA technologies, Coralville, IAcharacterization of the pluripotent state
Sequence-based reagentTDG1Hs02339499_g1IDT Integrated DNA technologies, Coralville, IAcharacterization of the pluripotent state
Sequence-based reagentTERTHs99999022_m1IDT Integrated DNA technologies, Coralville, IAcharacterization of the pluripotent state
Sequence-based reagentAPOBHs.PT.56a.1973344IDT Integrated DNA technologies, Coralville, IAcharacterization of the pluripotent state
Sequence-based reagentDHCR24Hs.PT.56a.4561516IDT Integrated DNA technologies, Coralville, IAexpression during guided cardiac differentiation
Sequence-based reagentELF4Hs.PT.56a.25941471IDT Integrated DNA technologies, Coralville, IAexpression during guided cardiac differentiation
Sequence-based reagentHN1Hs.PT.58.40922463.gIDT Integrated DNA technologies, Coralville, IAexpression during guided cardiac differentiation
Sequence-based reagentHSPG2Hs.PT.56a.18698732IDT Integrated DNA technologies, Coralville, IAexpression during guided cardiac differentiation
Sequence-based reagentHS6ST2Hs.PT.56a.1354985IDT Integrated DNA technologies, Coralville, IAexpression during guided cardiac differentiation
Sequence-based reagentLRP2Hs.PT.56a.1584067IDT Integrated DNA technologies, Coralville, IAexpression during guided cardiac differentiation
Sequence-based reagentMYLKHs.PT.56a.39795491IDT Integrated DNA technologies, Coralville, IAexpression during guided cardiac differentiation
Sequence-based reagentPCDH11XHs.PT.56a.26531358IDT Integrated DNA technologies, Coralville, IAexpression during guided cardiac differentiation
Sequence-based reagentPRTGcustom designIDT Integrated DNA technologies, Coralville, IAexpression during guided cardiac differentiation
Sequence-based reagentSIK1Hs.PT.58.2995158IDT Integrated DNA technologies, Coralville, IAexpression during guided cardiac differentiation
Sequence-based reagentSLC9A1Hs.PT.58.15072523IDT Integrated DNA technologies, Coralville, IAexpression during guided cardiac differentiation
Sequence-based reagentSDHDHs.PT.58.40267655.gIDT Integrated DNA technologies, Coralville, IAexpression during guided cardiac differentiation
Sequence-based reagentGAPDHHs.PT.45.8326IDT Integrated DNA technologies, Coralville, IAexpression during guided cardiac differentiation
Commercial assay or kitEdUClick-it Plus EdU Imaging KitLife Technologies
Chemical compound, drugBIO (GSK-3 Inhibitor)SigmaB1686
Software, algorithmPrism v7 and v8GraphPad Software

Additional files

Supplementary file 1

Differentially expressed genes between proband and parental 25 day old iPSC-derived cardiomyocytes.

https://cdn.elifesciences.org/articles/59554/elife-59554-supp1-v2.xlsx
Supplementary file 2

Gene function enrichment analysis for differentially expressed transcripts at day 25.

https://cdn.elifesciences.org/articles/59554/elife-59554-supp2-v2.xlsx
Supplementary file 3

Recessive and dominant genotypes identified in index case.

https://cdn.elifesciences.org/articles/59554/elife-59554-supp3-v2.xlsx
Supplementary file 4

Differentially expressed genes between proband and parental 0 day old iPSCs.

https://cdn.elifesciences.org/articles/59554/elife-59554-supp4-v2.xlsx
Supplementary file 5

Interactome of prioritized HLHS candidate genes.

https://cdn.elifesciences.org/articles/59554/elife-59554-supp5-v2.xlsx
Supplementary file 6

SKAT-O Analysis of 2 prioritized candidate genes in cases and controls.

https://cdn.elifesciences.org/articles/59554/elife-59554-supp6-v2.xlsx
Supplementary file 7

Rare, predicted-damaging LRP2 missense variants in cases and controls (CADD>24).

https://cdn.elifesciences.org/articles/59554/elife-59554-supp7-v2.xlsx
Supplementary file 8

Case-Control Association Analysis of Rare Variants in LRP2.

https://cdn.elifesciences.org/articles/59554/elife-59554-supp8-v2.pdf
Supplementary file 9

ENCODE Datasets.

https://cdn.elifesciences.org/articles/59554/elife-59554-supp9-v2.xlsx
Transparent reporting form
https://cdn.elifesciences.org/articles/59554/elife-59554-transrepform-v2.pdf

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)