Comprehensive multi-omics analysis of G6PC3 deficiency-related congenital neutropenia with inflammatory bowel disease

•Multi-omics approaches identify unique signatures•Whole-exome sequencing reveals distinct cytokine profiles•Expression of GATA4, PF4, and LST1 is dysregulated Autosomal recessive mutations in G6PC3 cause isolated syndromic congenital neutropenia which includes heart disease atypical inflammatory bowel (IBD). In a highly consanguineous pedigree with novel MPL, we performed comprehensive multi-omics analyses. Structural analysis variant MPL proteins suggests damaging effect. A molecular profile (cytokinome) the affected proband IBD was detected. Liquid chromatography-mass spectrometry-based proteomics G6PC3-deficient plasma samples identified 460 including 75 upregulated 73 downregulated proteins. Specifically, transcription factor GATA4 were while platelet 4 (PF4) upregulated. PF4 have been linked to respectively, may perturbed variety essential cell functions as it required for normal cell-cell communication. Together, these studies provide potentially insights into pathogenesis deficiency. Several familial syndromes can present severe neutropenia, or Kostmann syndrome, cyclic Hermansky-Pudlak warts, hypogammaglobulinemia, infections, myelokathexis Barth syndrome (X-linked dilated cardiomyopathy neutropenia), poikiloderma glycogen storage type 1b, among others (Banka, 2016Banka S. deficiency.in: Pagon R.A. Adam M.P. Ardinger H.H. Wallace S.E. Amemiya A. Bean L.J.H. Bird T.D. Fong C.T. Mefford H.C. Smith R.J.H. GeneReviews(R). 2016Google Scholar). The severity pattern nature complications, co-existing non-hematological phenotypes all assist making likely correct clinical diagnosis. Mutations several genes ELANE HAX1 (being most common) causes (SCN) (Dale et al., 2000Dale D.C. Person R.E. Bolyard A.A. Aprikyan A.G. Bos C. Bonilla M.A. Boxer L.A. Kannourakis G. Zeidler Welte K. al.Mutations gene encoding neutrophil elastase neutropenia.Blood. 2000; 96: 2317-2322Crossref PubMed Google Scholar; Klein 2006Klein Grudzien M. Appaswamy Germeshausen Sandrock I. Schäffer Rathinam Boztug Schwinzer B. Rezaei N. al.HAX1 deficiency autosomal (Kostmann disease).Nat. Genet. 2006; 39: 86-92Crossref Scopus (344) advent next-generation (NGS)-based mutation allows testing patients different etiologies. Consequently, more monogenic SCN are be diagnosed. (glucose-6-phosphatase, catalytic, subunit 3) initially 2 pedigrees (Boztug 2009Boztug Ashikov Salzer U. Diestelhorst J. Brandes Lee-Gossler Noyan F. al.A G6PC3.New Engl. Med. 2009; 360: 32-43Crossref (250) Since then, over 90 cases described, spectrum phenotype has reviewed Banka Newman, 2013Banka Newman W.G. review ubiquitous glucose-6-phosphatase caused by mutations.Orphanet Rare Dis. 2013; 8: 84Crossref (43) enzyme involved glycogenolysis, 3 known catalytic subunits being humans. encodes expressed ubiquitously humans, contrast G6PC1 (expressed small intestine) G6PC2 liver). maps chromosome 17q21 (GRCh38:44,070,699-44,076,343) consists 6 exons. manifest (i) non-syndromic SCN, (ii) so-called classic cardiovascular and/or urogenital abnormalities, (iii) form, Dursun involving non-myeloid hematopoietic lineages neonatal pulmonary hypertension thymic hypoplasia (Banka 2010Banka Özgül R.K. syndrome.Am. 2010; 152A: 2609-2611Crossref (30) 2009Dursun Ozgul Soydas Tugrul T. Gurgey Celiker Barst R.J. Knowles J.A. Mahesh Morse J.H. Familial arterial hypertension, leucopenia, atrial septal defect: probable new multisystem involvement.Clin. Dysmorphol. 18: 19-23Crossref (16) Inflammatory (like) reported some series (Bégin 2012Bégin P. Patey Mueller Rasquin Sirard Haddad É. Drouin Le Deist T lymphopenia deficiency.J. Clin. Immunol. 2012; 33: 520-525Crossref (33) Cullinane 2011Cullinane A.R. Vilboux O’Brien Curry Maynard D.M. Carlson-Donohoe H. Ciccone Markello T.C. Gunay-Aygun Huizing Gahl W.A. NISC Comparative Sequencing ProgramHomozygosity mapping whole-exome detect SLC45A2 single patient oculocutaneous albinism neutropenia.J. Invest. Dermatol. 2011; 131: 2017-2025Abstract Full Text PDF (56) Desplantes 2014Desplantes Fremond M.L. Beaupain Harousseau J.L. Buzyn Pellier Roques Morville Paillard Bruneau al.Clinical long-term follow-up 14 from French registry.Orphanet 2014; 9: 183Crossref (27) Glasser 2016Glasser C.L. Picoraro Jain Kinberg Rustia E. Gross Margolis Anyane-Yeboa Iglesias A.D. Green N.S. Phenotypic heterogeneity gastrointestinal illness associated founder mutation.J. Pediatr. Hematol. Oncol. 2016; 38: e243-e247Crossref (6) Hypo-glycosylation gp91phox, electron-transporting component NADPH oxidase, had demonstrated (Hayee 2011Hayee Antonopoulos Murphy E.J. Rahman F.Z. Sewell B.N. McCartney Furman Hall Bloom S.L. al.G6PC3 major defect glycosylation: mechanism dysfunction.Glycobiology. 21: 914-924Crossref (65) Failure eliminate phosphorylated glucose analog (1,5-anhydroglucitol-6-phosphate; 1,5AG6P) found (Veiga-da-Cunha 2019Veiga-da-Cunha Chevalier Stephenne X. Defour J.-P. Paczia Ferster Achouri Y. Dewulf J.P. Linster Bommer G.T. Van Schaftingen leads G6PT deficiency.Proc. Natl. Acad. Sci. 2019; 116: 1241-1250Crossref (31) Activating THPO also thrombocytosis predispose thromboembolism (Dasouki 2015Dasouki Saadi Ahmed S.O. THPO–MPL pathway bone marrow failure.Hematol. Stem Cell Ther. 2015; 6-9Crossref (3) Proteomics increasingly used search biomarkers many human diseases suggested protein signatures serum, feces, colonic epithelia (summarized Bennike 2014Bennike Birkelund Stensballe Andersen V. Biomarkers diseases: Current status identification strategies.World Gastroenterol. 20: 3231Crossref (66) Examples diverse include anti-Saccharomyces cerevisiae antibodies, perinuclear anti-neutrophil cytoplasmic antibody, C-reactive protein, calprotectin, lactoferrin, annexin A1, lymphocyte cytosolic 1 (aka L-plastin), proteasome activator 1, others. this study, extensive proteome analyses (with special emphasis on cytokinome) family members Southern Saudi Arabia (Figure 1) secondary (atypical) IDB thrombocytosis. We G6PC3, well multiple cytokines proteomic biomarkers. This complex multi-omic fingerprint help improve understanding mechanisms treatment opportunities. features results routine investigations summarized Tables 2. While failure thrive very low body mass index, only IV.6 diagnosed (atypical Crohn disease). Recurrent chest infections resultant bronchiectasis occurred siblings IV.1 but not their cousin (IV.6). All (atrial defect) requiring repair IV.6. Pan-T-cell two (IV.2, IV.6). Interestingly, serum IgE combination hyper-gammaglobulinemia family. However, no IGHE (which IgE) its receptors (FCER1A, MS4A2, FCER1G; FCER2) found. common MS4A2 FCER1B) previously thought atopy patients.Table 1Clinical characteristics individuals deficiency.Clinical manifestationsPatient IV.1Patient IV.2Patient IV.6InfectionsSkin chestSkin chestSkin; otitis media; recurrent E.coli UTI abdominal sepsisGIChildhood FTT, diarrhea/steatorrhea; symptoms adulthoodChildhood stricture diseaseRespiratoryBronchiectasis, class hypertensionBronchiectasis, respiratory failure, IV hypertension; lung transplantation candidateNot significantCardiacFenestrated ASD bidirectional flow (mainly left right); moderately RA, RV, main PADilated RV RVH, TR, raised RVSPASD repaired at age 8SkeletalGeneralized osteopenia; underdeveloped sinusesMicrocephaly, mid face hypoplasia; thoracic spine kyphosis; generalized osteopeniaOligoarthritis, osteopenia, facial dysmorphismGrowthWeight25.9 kg 1729.4 1617kg 20BMI1412.711.6Height<3rd centile<3rd centileASD, defect; BMI, index; thrive; IBD, disease; PA, artery; right ventricle; ventricular hypertrophy; RVSP, systolic pressure; tricuspid regurgitation. Open table tab Table 2Routine laboratory investigationsLaboratory resultsPatient IV.6Neutrophil count x109/L, Median (range)Pediatric: 0.5 (0.11–2.25)Pediatric: 0.31 (0.09–1.76)Pediatric: 1.39 (0.8–6.2)Adult: 0.34(0.24–1.77)aInitial (pediatric) done early childhood (3–5 years age). Follow-up (adult) between 17 age.Adult 2.385 (0.4–4.57)aInitial age.Adult: 1.65 (0.67–17.2)Lymphocyte median (range)2.95 (1.59–3.27)2.4 (1.2–8.5)0.49 (0.14–2.17)Platelet counts, (range)498 (344–777)392 (179–738)498 (150–1074)Hb g/L, (range)108 (84–130)100 (77–127)97 (58–147)Bone marrowPediatric: Unremarkable maturation arrestPediatric: marked left-shifted granulopoiesis active erythropoiesis megakaryopoiesis–Adult: hypoplastic myelopoiesis shift abnormal segmentation myeloid precursorsAdult: hyperplasia, shifted minimal increased granulationAdult: Left myelopoiesisBone karyotype46, XX46, XXIgA (normal: 0.7 - g/L)1.92.931.72IgG 7–16 g/L)27.856.623.3IgM 0.4-2.3 g/L)0.630.772.5IgE 5–500 KU/L)<2<22.5T-cell subsetsNormal T-cell subsetsT-cell lymphopenia; intact expression MHC I Il antigensAbsolute CD3+, CD4+, CD8+ lymphopeniaa Initial age. ASD, Sanger CFTR SBDS negative. Pathogenic pathogenic variants patients' exomes listed 3. homozygous missense exon (c.479C > T, p.Ser160Leu) confirmed segregation 1). state various public databases dbSNP, 1000G, ESV, ExAC, gnomAD, our private Genome Program database (https://www.saudigenomeprogram.org/). Only (African) heterozygous individual (allele frequency: 0.000004061) recently gnomAD (https://gnomad.broadinstitute.org/).Table 3Pathogenic cytokinome profilesGeneaDisease-gene association data based OMIM “www.omim.org”. (mutation/variant)Disease associationaDisease-gene “www.omim.org”.Patient IV.6G6PC3:NM_138387:exon4:c.479C T:p.S160LSCN4 (AR), syndromehmzhmzhmzMPL:NM_005373:exon4:c.398C T:p.P133LCAMT thrombocythemia (AD), MFMM (som)wtwthetHBG1:NM_000559:c.6_aDisease-gene “www.omim.org”.3delinsCTCTHbF (QTL1)hmzhmzhmzKLF1:NM_006563:exon2:12996130-12996956:-14:840HPFH CDA 4wtwthmzMS4A2 (FCER1B): NM_001256916:exon6:c.575A G:p.E192Gsusceptibility atopyhetwthmzCCL15:NM_032965:exon1:c.5A T:p.K2MNonehmzhmzhmzCXCR2:NM_001557:exon3:c.1075A T:p.T359SNonewthethetIFNAR1:NM_000629:exon8:c.1143 + 12G ANonehetwtwtIFNAR2:NM_000874:exon7:c.611C G:p.T204RIMD45 (AR)hetwtwtIFNW1:NM_002177:exon1:c.247A G:p.M83VNonewtwthetIL10RA:NM_001558:exon1:c.67 11G CIBD28 (AR)hetwthetIL16:NM_172217:exon4:c.643T C:p.S215PNonehetwthetIL18R1:NM_003855:exon6:c.626-7C TNonehetwthetIL1R1:NM_001288706:exon12:c.1211-4T CNonehetwthetIL1RL1:NM_016232:exon11:c.1501_1502delinsAGNonehetwthetIL21R:NM_021798:exon9:c.1381dupG:p.A460fsIMD56 high (AD)hetwtwtAD, dominant; AR, recessive; CAMT, amegakaryocytic thrombocytopenia; CDA, dyserythropoietic anemia; CCL15, chemokine, cc motif, ligand 15; CXCR2, CXC receptor 2; 3; HBG1, hemoglobin gamma A; het, mutant; hmz, HPFH, hereditary persistent fetal hemoglobin; IMD, immunodeficiency; IFNAR1, interferon-alpha-beta- omega 1; IFNAR2, IFNW1, interferon, omega-1; IL10RA, interleukin 10 alpha; IL16, 16; IL18R1, 18 receptor-1; IL1R1, IL1RL1, receptor-like IL21R, interleukin-21 receptor; KLF1, Kruppel-like MFMM, myelofibrosis metaplasia; myeloproliferative virus oncogene; QTL, quantitative trait locus; neutropenia; som, somatic; wt, wild type.Cytokinome refers patient-specific rare DNA variants.a Disease-gene AD, type. Cytokinome variants. Also, c.154C (p.Arg52Cys) MBL2 observed IV.1. silico predicted Caucasians (Choteau 2016Choteau L. Vasseur Lepretre Figeac Gower-Rousseau Dubuquoy Poulain D. Colombel J.-F. Sendid Jawhara Polymorphisms mannose-binding lectin defective functional activity Crohn’s patients.Sci. Rep. 6: 29636Crossref (9) Additionally, exome (but her cousins 2) showed 3.255 × 10−5), possibly (Chr1:43804948:43804948: NM_005373:exon4:c.398C p.P133L) intermittent thrombocytosis, detected HBG1 (NM_000559: c.∗6_∗3delinsCTCT) addition another splicing KLF1 (NM_006563:exon2:12996130-12996956:-14:840; GC −) probably explains elevated HbF levels patients. 346-amino acid contain nine transmembrane helices that span endoplasmic reticulum (ER) membrane, site lying inside ER lumen (Ghosh 2004Ghosh Shieh J.-J. Pan C.-J. Chou J.Y. Histidine 167 phosphate acceptor glucose-6-phosphatase-β forming phosphohistidine intermediate during catalysis.J. Biol. Chem. 2004; 279: 12479-12483Abstract (44) Prediction 3D structure threading templates such crystal PAP2 phosphatidylglycerol phosphatase Bacillus subtilis (see transparent methods) revealed Ser160 located close end fourth helix, near luminal side. contact neighboring helix buried hydrogen bond intramolecular interactions Arg161, Thr30, backbone carbonyl Trp26 2, G6PC3-A, B). Thus, plays an important role tethering together. Ser160Leu would disrupt interaction network steric clashes G6PC3-C). Hence, destabilize interface thus affect tertiary region, could reduce efficiency coupling transporter 6-phosphate translocase (SLC37A4). amino residue Pro133 extracellular domain contains homology regions (CHRs), each composed fibronectin III (FNIII) domains. part linker connecting first second FNIII N-terminal CHR (CHR1). CHR1 region inferred ∼28% sequence identity erythropoietin 3, MPL-A). Computational modeling substituting larger hydrophobic leucine affects stability dynamics MPL-B). Given relative orientation domains critically signaling through (Syed 1998Syed R.S. Reid S.W. Li Cheetham J.C. Aoki K.H. Liu Zhan Osslund Chirino A.J. Zhang al.Efficiency depends orientation.Nature. 1998; 395: 511-516Crossref (459) Scholar), Pro133Leu expected perturb activation signaling. Peripheral blood subjects causing IV.6, healthy subjects, analyzed using liquid chromatography-tandem spectrometry (LC-MS/MS). total species (Table S1) 148 significantly differentially (≥2 ∞: fold change p < 0.05) deficient control S2). Classification those biological processes groups immune system, homeostasis, molecule transport, vesicle-mediated respectively (Figures 4, 5, 6). 21-protein subpanel represented networks ingenuity (IPA, Qiagen) 7 8, respectively) STRING tools 9). annotations profiles other detailed Figure 5.Figure 5Biological process classification CSN samplesView Large Image ViewerDownload Hi-res image Download (PPT)Figure 6Differential deficiencyShow full captionHeatmap (CSN) controls. Patient controls pooled separately run triplicates.View 7IPA captionIngenuity Pathway Analysis (IPA)-based panel. displayed graphically nodes (gene/gene products) edges (the relationship nodes). node color intensity indicates genes: red green plasma. values indicated under corresponding node. Node shapes indicate product shown key.View 8IPA 9The inter-relationships 21 mapped STRINGS databaseShow captionSome molecules function enzymes, coagulation factors, transporters, regulators, homeostasis differentiation regulators. Others act kinases, peptidase growth cytokines. (The done, above figures generated Ingenuity program [IPA, version 8.7] string, respectively). AGT, angiotensinogen; AKT1, AKT serine/threonine kinase BCL2L11, BCL2-like 11; C1QA, complement q subcomponent, chain; C1QB, B C2, F9, IX; FOS, FOS protooncogene; FOSB, FOSB FOSL1, FOS-like antigen FOSL2, HBA1, hemoglobin-alpha locus HBB, hemoglobin-beta IL6, 6; IL6R, JUND, oncogene JUN-D; MCL1, leukemia PIK3CG, phosphatidylinositol 3-kinase gamma; REN, renin; SERPINC1, serpin inhibitor clade C (antithrombin) member 1.View (PPT) Heatmap triplicates. key. Some conn