MM, MM, MAF, DA, and AM contributed equally to this study. [email protected] Email: 2406. 2249 965 + Fax: 4343, Ext. 2999 2224 +965 Phone: Tel: Institute, P.O.1180, Box Dasman15462, Kuwait. This articlehas beenaccepted for publicationundergone and full peer review buthas not been author: Corresponding 1 * MelhemMotasem ANEMIA AND title: Running thrombocytopeniaand anemia recessive autosomal causevariant familial G6B Novel Title: Article :Article type Original Article 07-Oct-2016 : Date Accepted 16-Aug-2016 : Date Revised : 30-May-2016 Date Received This article is This protectedAll rights byreserved. copyright. 10.1111/ejh.12819 Version the and version this between differences throughcopyediting, the typesetting, pagination and proofreading process, which may leadto & transplantation Unit, Dasman Diabetes Institute, Kuwait; Kuwait; Institute, DasmanDiabetes Unit, transplantation & L Beales L Hospital, Canada. Hospital, Genomic Medicine, UCLGenomic Health, Child of Institute London, UK; Dasman Diabetes Institute, Kuwait; Dasman Kuwait; Diabetes Institute, and Molecular Biology Department, Biology and Molecular Chiara Bacchelli Genetics andGenomics Department, Genetics Accepted Contributed equally Article 3 , and Osama Alsmadi Osama and , Melhem et al. G6B NOVEL VARIANT CAUSETHROMBOCYTOPENIA etal.G6B VARIANT NOVELMelhem 3 , Fadi Alkayal

1* , Mohamed Abu-Farha Mohamed , Dr. Osama Alsmadi, Genetics and Genomics Unit, 4 1 , Irina AlKhairi , Irina

Dasman Diabetes Institute, Kuwait; Kuwait; Diabetes Institute, Dasman 6 Department of Pathology, Windsor Regional Regional Pathology, Windsor of Department

Dasman Diabetes Institute, Kuwait; Kuwait; Institute, Diabetes Dasman of Record. Please cite this article as doi: asdoi: article this cite Please Record. of 2* , Dinu Antony , Dinu 2 , SumiJohn 5 5 Integrative Informatics, Integrative Informatics, , Mohamad Alomari 1* , Ashraf Al MadhounAshraf , 4 Pancreatic islet Biology Biology islet Pancreatic

Dasman, Diabetes Diabetes Dasman, 3 Genetics and and Genetics 2 Biochemistry 6 , Phillip 1* , Anemia, platelets, thrombocytopenia, G6b, C6orf25 G6b, thrombocytopenia, platelets, Anemia, Key Words G6b from reported were outcomes similar where by in evidence vivo supported further well disorder.is This inference hematological observed for the as gene responsible the and regarded this family, in transmission Conclusion and SHP-2. SHP-1 phosphatases 2 domain-containing region Srchomology regulators, hematopoiesis key the for as C6orf25, also known and erythrocytes. megakaryocytes into K562differentiation enhanced in expression resulted G6B wild-type form, the truncated Unlike G6B theprotein. encodedtruncated destabilizing wasfor responsible mutation, 25( Frame Reading Open Results gene. defective andcharacterize the toidentify out carried Methods (low platelets count) and anemia. bloodthrombocytopenia disorder with with manifested life-long family consanguineous Objective Abstract This article is This protectedAll rights byreserved. copyright. Accepted Article : We identification of a novel truncation mutation (p.C108*) in 6 6 Chromosome in (p.C108*) mutation truncation novel a of identification : We

: Genetic linkage analysis, exome sequencing and functional genomics were genomics were and exomesequencing functional Genetic analysis, linkage : : To characterize the underlying genetic and molecular defects a in and genetic molecular underlying the To characterize : : G6B seemsto act through diseaserecessive an autosomal mode of

C6orf25 ) gene in this family. We alsop.C108* showed the family. this We gene in )

-/- and SHP1/2 DKO mouse models. models. mouse andSHP1/2 DKO

G6B , is an effector factor erythroid 2( erythroid factor ( levels normal below are that counts platelets by low characterized Thrombocytopenia is anomalies.blood these of examples distinctive areanemia two and Thrombocytopenia differentiation. and/or division cellular abnormal of aresult as cells progenitor marrow thebone molecular impairments in developmental from result can disordersBlood Introduction This article is This protectedAll rights byreserved. copyright. Thalassemia, and both are expressed as recessive traits [15]. There are no data data no are [15].There traits recessive as andboth expressed are Thalassemia, anemiaare cell and mainly sickle hemoglobinopathies world, [14]. In the Arabs worldwide disorders hemoglobin recessive inherited of themajority constitute anemia andsickle cell 13].Thalassemia anemia heritable andalso [12, are sickle cell Fanconi thalassemia, including anemia of forms Certain [11]. X-linked or recessive, dominant, asautosomal betransmitted may thrombocytopenia 10].Heritable [9, formation ( oncogene FLI1 example proto overfor other; one the lineage of generation thealter can Other factors [8]. count, respectively andplatelets anemia in low which manifest ( 1 leukemia lymphoblastic acute cell T- factors such as transcription in other [7].Defects and/or thrombocytopenia anemia to defectsgene canlead GATA1 in Genetic 1 (GATA1) [6]. protein binding GATA including factors transcription by regulated several aprocessin is that progenitor cells ancestor acommon from RBCs derive and (MEGs) megakaryocytes producing counts Platelets- (RBC) [1-5]. cell blood red low and/or contents hemoglobin in ˂ FLI1

Accepted Article50,000/ ) prompts MEGs production, whereas Kruppel-like 1factor ( whereas Kruppel-like production, MEGs ) prompts μ l) compared to healthy individuals, whereas anemia is caused by a reduction areduction by causedis anemia whereas compared individuals, tol) healthy NFE2 ), can also lead to erythroid and megakaryocytic alteration, alteration, can also), and megakaryocytic to erythroid lead TAL1 ), FOG familymember 1(

KLF1 FOG1 ) prompts RBCs RBCs prompts ) ), andnuclear β - the characterizing and identifying for undertaken schemes differentiation G6B enhance this of to failed thetruncated form erythrocytes; and into megakaryocytes whichdifferentiation K562 cells enhanced expression mutation was G6B in by thisresponsibleas phenotype, the evidenced for showplatelets phenotype. countswith combined reduced anemia that atruncation We life-long exhibiting are significantly affected members family. All Arab consanguineous a defective of thediscovery we sequencing, report whole exome and mapping homozygosity Using gene deleterious variants. embracing are that (ROH) homozygosity of regions of enrichment the from result direct manifests withrisk autosomal increased of recessivedisorders thisincrease [18]; isa naturally 0.125, and that of (F) co-efficient breading cousinswith first double unites practice often this inbreeding families, Arab some Mediterranean For countries [17]. some for marriages all of 50% exceeding East, Middle the in practiced commonly Marriage [16]. world are marriages overall between relatives consanguineous is the% of study 10.4 such A2010 reported in of disorders. the transmission dominating rareincidence the of disorders, and with recessive autosomal heritable forms increased with associated are size family large with combined rates consanguinity High current time. at the amongArab population thrombocytopenia onthe heritable available incidence of This article is This protectedAll rights byreserved. copyright. Accepted Article G6B G6B recessive novel in gene multiplyaffected siblings from a

G6B gene are discussed. gene

wild - type G6B . The The exomeanalysis. via sequencingand analysis tier the second in candidate of shortlisting the guide wasused to analysis, Golden Helix softwarewasutilized analysis for the [19]. locus The critical thefrom FBAT using testbased consanguineous association Family family. (FBAT) penetrance this in 100% with transmission, model autosomal disease an of recessive assumed analysis Linkage software. GenomeStudio Illumina checks, (QC) using control and quality concordance for wasused 99.9% of rate Call Kuwait. centre, genome Institute Diabetes Dasman out in was carried Genotyping SNP markers). n=730,525 USA; CA, Diego, San Inc.; (Illumina array genotyping bead OmniExpress Illumina using gene, disease the harboring locus the identify to performed was approach genotyping density High Analysis Linkage andDNAextractions. RNA plasma, for tubes anti-coagulated EDTA blood of two 8ml into of withdrawal by followed historyjoined this study.was Clinical taken for all members by doctor, family their voluntarily and agreed participants All origin. anArabian of consanguineous family a and belong21 to adults theareage years, above all Study participants Participants of Study Enrollment Methods: This article is This protectedAll rights byreserved. copyright. Accepted Article

For transient transfection assays, 10X10 assays, transfection For transient G6B serum and wild-typelength) penicillin/streptomycin. Both and(full the oftruncated forms bovine fetal 10% with supplemented (EMEM) Essential Medium Minimum Eagle’s using suspension in cultured were Cells MD). Baltimore, (Rockville, Collection Culture Type American from obtained was line (K562) cell leukemia myelogenous Human K562Cells using Transfection Transient Plasmids G6B 2 1 & Tables settings filter Detailed supplementary are given in protocol. standardized Enrichment TrueSeq out Illumina Exome was according to sequencing carried For Whole Whole Exome Sequencing This article is This protectedAll rights byreserved. copyright. Accepted respectively, as [20, treatments, described butyrate (Na-Bu) (PMA)and sodium acetate 12-myristate13- cells using and phorbol erythrocyte-like platelet-like into differentiated were constructs, G6B described the with cells, K562 transfected Transiently cells K562 of differentiation Erythrocyte-like versus Platelet- analysis. protein for harvesting before 24-hours another for media EMEM complete in grown were cells and the transfection of 24hours after was Media media. changed free antibiotics in cells the to andadded CA) Carlsbad, (Invitrogen, manufacturer by the recommended 24 used. Briefly, or wild-type either truncated with mock, Article were cloned into pCMV-6 vectorwith either FLAG or GFP tags at thec-terminus. .

μ g of plasmid DNA were mixed with Lipofectamine 3000 as as 3000 Lipofectamine with mixed were DNA plasmid of g G6B 6 K562 cells were used and were transfected transfected were and used were cells K562 . Both FLAG and GFP tagged clones were

21] This article is This protectedAll rights byreserved. copyright. Accepted and levels toVI havethe lowest relative VII. bothhemoglobin to happened and splenomegaly asignificant developed and V II Interestingly,respectively. siblings years, and 28 30 41, 38,33, 48,45,42, are 50, youngest (IX) oldestto (I) from siblings Ages of abnormalities. cousins,hematological noare with of first history parents The and anemia. thrombocytopenia early with childhood at persisting manifested Vand (II,IV, VII) of the siblings was thisstudy. brothers)seven in Four investigated and (two daughters siblings with nine 1) (Figure family consanguineous Arabian A Findings Microscopic and Summary Clinical Results (*P s.e.m. error variance is and assumingt-test equal with Student’s aone-tailed estimated was significance Articleexperiment. Statistical within each variations normalized to reduce where efficiencies transfection The experiments. independent three in differentiated and transfected transiently K562 triplicates. in performed wereexperiments (6hrs,differentiation days extractions.2, 4, course All 6) for mRNA and intervals a were harvestedat time PMA.Cells of instead with 0.6nMNa-Bu containing media a were differentiation K562 cells treated generation, the transfected erythrocyte-like for Similarly, days. 6 of period a for daily changed was media differentiation The PMA. nM and 16 FCS,1%Pen/Strep, RPMI media a 10% containing were differentiated typewith transfected transiently wild G6BG6B, truncated or vectorempty thenand

< with minor modifications. For platelet-like differentiation, K562 (5x10 differentiation, platelet-like For modifications. with minor 0.05). 0.05).

5 cells) were were cells) bone marrow because of the dry tap. Therapeutic interventions were limited to providing toproviding limited were interventions Therapeutic thedry tap. of because bonemarrow on the was performed karyotypes. karyotyping No normal studies showed Cytogenetic myelofibrosis. with consistent disorder myeloproliferative with this in family, featureswith most compatible is phenotype the determined and all hematopatholgists, independent by evaluated were CD34 alsoidentified.A moderate was reticulin tosevere fibrosis also present.No increase in were megakaryocytes enlarged Some clustering. focal with number in increased were Megakaryocytes represented. being threecell lines well with hematopoietic all cellular hyper- to normo-cellular were smears biopsies The tap. dry a showing these all with course, bone life biopsies, marrows doneovertheir hadmultiple Each sibling affected reaction). be (leukoerthroblastic could identified also and metamyelocytes) myelocytes (e.g. precursors and neutrophil blood cells red nucleated Rare drop shape. with showed tear anisopoikilocytosis blood giant red moderate cells The platelets. of numbers platelets except increased normal was of within The for morphology siblings. the and toconfirmed compared morphology, severe the normal thrombocytopenia white cells blood normal affectedshowed blood those of smears The peripheral reported. was either immunity decreased of signs or infections increased of history No not. of affected whether siblings all for normal always were counts cells blood White bleeding. major of history no is 10,000-40,000/ withcounts from platelets ranging siblings affected wasalso the in observed Variable thrombocytopenia gm/dl. 10 6 and between ranging life their throughout varied participants affected the of levels Hemoglobin This article is This protectedAll rights byreserved. copyright. Accepted Article + blasts could be demonstrated. The bone marrows and peripheral blood smears andperipheral blasts bonemarrows couldbe demonstrated. The

μ l. Apart from bruising, therefrom Apart l. Results revealed an FBAT log anFBAT Results revealed Sequencing Exomes and Analysis Linkage improvements. any to led not had this however levels, hemoglobin lower with Treatment steroids was attempted with the two showing siblings splenomegaly and acid werewhen needed. given transfusions Occasional supplements.iron and B12/folic This article is This protectedAll rights byreserved. copyright. Accepted 6, chromosome on interval linkage the to mapped 44 variants, 2571 the Of family. this in detected were deleterious 2571 variants of genomic total 3). A Table (Supplementary were catalogued &rs1537638 rs13195345 SNPs between the linkage interval critical within the identified deleterious variants List of details). parameters analysis 2 for 1 and software tables was (see this analysis for supplementary analysis used variant Ingenuity unaffected normal of controls. our database 40exomes from with along in-house respectively),V in addition to anunaffected male (III) from this familywere sequenced, &was affectedsister andhis (II one male on affected exomes Analysis carriedfrom out States). United MA, Institute, Broad (IGV; Viewer Software Genomics were assessed using Integrative reads the sequence of quality The 1. Table supplementary shown in is analysis ingenuity for cascade used Thefilter analysis. assumed for was inheritance of mode recessive an autosomal levels platelet and RBC normal andparents had both were affected membersof gender Since genefamilies. Articleautoimmune amongstHLAorthe classified which locus, of genes this weremany in bands.500 than present more cytogenetic There which rs13195345 & encompass rs1537638, a between p22.3 region and p21.1 6, between SNPs chromosome of arm “P”short petite on the stretch 27.7Mb DNA -10 -10 score = 1.94, with the critical interval minimized to interval minimized thecritical score1.94, with =

(Y211 and Y237). Y237). and (Y211 (ITIM) with motif tyrosine-based inhibition its twoimmunoreceptor tyrosine residues the encompasses which domain cytoplasmic the to addition in domain, transmembrane the eliminates mutation This (p.C108*). TGA codon stop with the peptide encoded position the 108 in the cysteine substituting codon at mutation nonsense a truncation to corresponds c.324C>A 2. Figure in shown is family the in genotypes and mutation the showing Arepresentative chromatogram sequencing exome project andExAC. NHLBI including project,variantabsent the dbSNP,1000 global genome databases from rare was this variant also Furthermore, novel variant anddeleterious. is this confirming 94 ethnically-matched from another again healthy unrelated DNA controls controls, when in examined was absent variant c.324C>A also sequencing confirmed Sanger consanguineous disease this autosomalin pattern Mendelian transmission family. whichwith consistent a (see is 1), siblings figure andclassical unaffected deceased) (father mother the in washeterozygous variant this whereas siblings, affected the familyparticipating genotypes members homozygous (The confirmed as were only in eight all via Sangersequencing genotypes in examining its by (segregation) The identified gene this predicted alteration. effect pathogenic from the confirming further 1485, of and anodds ratio associationof p=4.71E-07, showed an value controls. the40 in-house of anyor in family, affected siblings. This changewas not seen in the unaffected (III) sibling thisfrom and only This article is This protectedAll rights byreserved. copyright. Accepted Article C6orf25 C6orf25 (c.324C>A; p.C108*) was aseen two state was in homozygous p.C108*) (c.324C>A; in the variant (c.324C>A) was assessed further for its clustering clustering its for further was assessed (c.324C>A) variant

p.C108* according to ingenuity analysis, p.C108* to ingenuity according

cells, the transcription of of thetranscription cells, these In differentiation. platelets of stimulation asignificant showed G6B wild-type with transfected cells treated PMA K562 Interestingly, . 25] [24, 3A) (Figure differentiation suggestingcontrol plasmid; sufficient PMAis to induce megakaryocyte/platelet (mock) the scrambled with transfected that are cells transiently undifferentiated to the respectively, relative CD61, the CD41 and inductionof platelet markers unique 320-fold and 33- demonstrated analyses qrtPCR K562 differentiation, 6 of day At respectively. Na-Bu, or PMA of presence the in K562, line cell leukemia myelogenous chronic was evaluated platelet or usingprecursors, the human erythrocytes differentiation circulation [22, 23]. blood the in detected canbe that andGolgi reticulum endoplasmic the found in transcript different this of variants gene that also encode can solubleisoforms protein be however membrane, plasma the to bound is that receptor surface cell glycosylated a is gene by this encoded protein The (Ig) superfamily. the immunoglobulin of member 6,region III onchromosome anda (MHC) class complex histocompatibility major C6orf25 This article is This protectedAll rights byreserved. copyright. the truncated G6B revealed statistically insignificant insignificant G6B statistically the revealed truncated with transfected hand, cells other lineage. On the platelets of early induction enhances of expression the these cells, in construct. Further, plasmid (mock) the scrambled with transfected cells Accepted megakaryocytes, of in theenhancement G6B wild andtruncated of type The role Article differentiation lineage hematopoietic enhances G6B gene, also known as CD41 and CD41 CD61 G6B and and (Ensembl ID: ENSG00000204420), is located the in is ENSG00000204420), ID: (Ensembl was atday significant highly G6B 4; implying that CD61 was 33- and22.5- higherfolds of than that

CD41 and and CD61 expression (at 1- 1- (at expression 6 plasmid vector constructs. Western analysis on cell lysates withG6B specific oncell analysis vector constructs.6 plasmid Western pCMV- using were FLAG-tag expressed K562 cells in of theprotein forms and truncated wild-type both on G6Bprotein, the of the mutant truncation effect tostudy the order In Truncated Truncated not shown). (data lineages hematopoietic Bu, respectively. Nevertheless, G6B not wasalone to sufficient induce the K562 of cells orinto or inPMA of erythrocytes, platelets presence the Na-differentiation 3B). (Figure significant statistically and respectively, higher, 8.5-folds and 11.6- was vector scramble both 6, day At 4. day at starting induction relative a substantial with expression genes the erythrocytic enhanced particularly G6Bwild-type transfection, G6B 3B).The expressing truncated the (Figure cells observed in also was magnitude and trend same The [21]. report previous expressiontorelative baselineday 0,re at and 6-folds 15- with and KLF1, CD44 markers oferythrocyte expressions temperate showed cells K562 plasmid-transfected the scrambled conditions, differentiation 6under similar day and lineage. At tothe erythropoetic specific markers of expression 3A). (Figure cells G6Bin these endogenous the role for possible functional a residual suggesting transfections, plasmid with to that seen the scrambled respectively), similar and 2-fold, This article is This protectedAll rights byreserved. copyright. Acceptedenhancing the in G6B for role potential a revealed results these Takingtogether, Articledifferential distinctive in resulted Na-Bu with treated cells K562 of Transfections G6B Leads to Degradation Degradation to Leads CD44 and KLF1 relative expression, compared to that of the the of that to compared expression, relative spectively (Figure 3B), consistent with awith consistent 3B), (Figure spectively

Both wild-type and wild-type Both truncated forms of 4B. asshown Figure efficient was in equally byboth constructs transfection confirmed of analysis QuantitativeRNA 4A). rtPCR itsabsent, degradation (Figure suggesting was form theG6Btruncated is detectablewhile wild-type protein showed antibodies This article is This protectedAll rights byreserved. copyright. the truncated form Figure 4E. form truncated the to did stabilization any notcause G6B and expression affect thewild-type protein not did with inhibitor the lysosome Treatment 4C. Figure form truncated the of stabilization notthedid but caused wild-type, affect mild with inhibitor theproteasome Treatment 10 (MG132, inhibitor or expressingcells G6B either withtruncated were wild-type treated proteasome G6BwhetherToby the degraded protein test lysosome is or proteasome mechanism, Protein G6B Truncated the Stabilize Tagging and GFP Inhibitor Proteasome control. tothecompared mock Accepted F. and D, 5B, Figure in shown as inhibitors truncated and wild-type G6B and was not affected by the proteasome or the lysosome E. 5C, Figure in shown as form either with the proteasomeor the lysosome inhibitors did not affect the protein stability for 5A.Treating Figure in as shown form the of truncated stabilization had led to protein G6B of thetruncated GFP tagging our surprise, protein. To theG6B of expression thetagon outof the impact G6B was to rule used GFP also protein tagged F. 4D, Figure treatment either bywas notaffected Article μ M) and lysosome inhibitor (chloroquine, 100 (chloroquine, inhibitor lysosome and M) G6B gene expression for both treatments was similar and and similar was treatments both for expression gene G6B G6B showed over 800 fold increase in expression expression increase in 800 fold showed over gene expression was similar wasbetween gene expression similar

μ M), respectively. mutation (c.324C>A) in the in (c.324C>A) mutation have ahomozygous identified characterized gene. notawell [23],We I &II classes is 6p21.3). novel adefective identified we and have anemia, with thrombocytopenia manifested disorders blood unusual with family a consanguineous on exomesequencing and analysis linkage Using Discussion Figure in 6 C- D. itsin structureas possiblycausedchanges stabilization toits shown has lead major that On theof wild-type. hand,tagging the other withG6B truncated GFP protein has of G6B is missing the beta-sheets form in present structure the truncated structure. The G6B protein contains structure of a number anbeta-sheets forms that organized tertiary G6B. of Asshownforms andtruncated wild-type predicted 6A,the Figure in wild-type the of structure [26], wepredicted protein the Using SWISS-MODEL the Workspace Structure protein G6B Predicted This article is This protectedAll rights byreserved. copyright. Accepted (MP-Shp1/2 DKO) knockoutdouble mouse models SHP1/2 conditional Furthermore, and megakaryocytic production, function. development, platelet29] affecting andSHP-2 [28, SHP-1 of regulator playsas major and through, suggested G6Bsignals knock mousewith [27]. out data Recent SHP-1 also andSHP-2 interaction through the of aggregation platelets humanon its G6B we primarily in inhibition role is What know causal. that thismutation is homozygous withsuggestingof the cosegregation c.324C>A strong disease is proof Article G6B , alsoas known, G6B G6B C6orf25 gene which maps to short arm of (band (band 6 chromosome of arm short to maps which gene gene, only in the affected members of this of family, members only affected gene, in the , which, localizesMHCIII locus, between to MHC

as intracellular signaling transducer [32, 33]. LYN is a major player in cell cycle cell in player amajor is LYN 33]. [32, transducer signaling as intracellular act andhence catalytic motifs, own their who lack receptors with membrane associated physically is and proliferation, lineage myeloid the in role inhibitory a known with [32] family SFKs belonging tothe kinaseseight the oneof is LYN (SFKs). kinases family G6B LYN,also of phosphorylation underof theis of The the amember Src control [30, 31]. mechanism are recruited the negatively to regulate activation signal via a dephosphorylation activation G6B, SHP-1and/or Upon respectively theof SHP-2 [31]. membrane-bound by encodedare SHP1 &SHP2 Human andSHP-2. SHP-1 tyrosine (PTPs) protein phosphatases such 2as homolog domain-containing (SH2) with Src tointeract areknown ITIMs tyrosine [30]. residues two with (ITIM) motif inhibitory tyrosine-based cytoplasmic a and segment, atransmembrane with receptor G6B studies. their was documented in such phenotype uncertain if we are and production, erythrocytes on no information was mice there model, count G6b-B–deficient in areduced platelets reported colleagues [29] and Mazharian function. While of loss G6B their for compensating independent, G6B- thatis aexists mechanism, redundant whether andquestion intriguing it we find mutation, p.C108* the for homozygous are who members family affected our of Incase life. of weeks few the first in observed as model mouse this in was lethal DKO [28].SHP1/2 hematopoiesis defects andimmunity in significant highlighting cells, blood white in increase 4.2-fold a and cells blood red in reduction 26% a including aberrations, additional hematologic addition an production, had to severely platelets reduced in This article is This protectedAll rights byreserved. copyright. Accepted Article encodes a putative cell surface immunoglobulin (Ig) superfamily membrane membrane superfamily (Ig) immunoglobulin surface cell putative a encodes

PTPN6 and and PTPN11 genes, (figure 3 A&B). is (figure 3A&B). It reasonable to incasespeculate that, family, of our this whole G6B mutant or wild-type the of presence in differentiation driven Na-Bu- and PMA- our by supported is This genes. regulating theabove from that independent is mechanisms pathological a propose and further G6B protein, expressing anon-functional in of this athe affected observed ofmembers is consequence family, phenotypes hypothesize and NFE1, NFE2, [38]. and FOG1 thatanemia thrombocytopenia We GATA2, GATA1, including pathway differentiation this in shared are factors transcription (HPCs).Several progenitor cells hematopoietic acommon from precursor event from differentiation a late through are generated erythrocytes and megakaryocytes Both [37]. respectively anderythrocytes, platelets of tothe formation lead subsequently which into a differentiation precursormyeloid of megakaryocyte and erythroblast lineages and proliferation including effects cellularsignaling cascadedownstream triggers LYN mouse studies. with the consistent which is here, studied family the in reported complications that were one thewassplenomegaly of clinical tumors [32]; macrophage and monocyte of types several and splenomegaly, including phenotypes of spectrum a studies. LYN-deficient display mice mice derived is from kinase knowledge on this the and mostof current literature, in available data is human LYN Only scarce 36]. [35, theactivation cascade of part as recruited also are (PI3K) kinase inositol-3 phosphatidyl C phospholipase (SYK), tyrosine spleen kinase kinaseslike tyrosine of G6B), within the (e.g. receptor by ITIM residues LYN of other phosphorylation [34]. Upondivision cells in required is phasewhich G1 into entry their trigger cells these on receptors immunoglobulin of activation The B-cells. in signaling This article is This protectedAll rights byreserved. copyright. Accepted Article

γ 2 (PLC γ 2) and and 2) Conflict- of interest disclosure: The authors declare no competing financial interests. financial authors no competing declare of interest The disclosure: Conflict- manuscript. the anddeveloped data interpreted study, results; designed O.A of discussion to contributed investigations; P.L.B. clinical M.A.carried out analysis; out FBAT carried S.J. carriedperformed analysis; I.A. outwesternarray genotyping; F.A. bead analysis., data exome writing manuscript; in performed C.B. andcontributed data, interpreted D.A. assisted A.M. and M.A.F., and& designM.M., analyzed research, in experimental Contribution: Authorship authorsAll of no declare conflict interest. science. of Advancement for Foundation and Kuwait Institute Diabetes by the Dasman funded was study research This study. this in participating for members family all thank We Acknowledgments pathogenesis. andthrombocytopenia anemia in exact its role delineate in to , and further this gene’s dissect mechanism molecular be essential FutureG6B investigations onG6B usingKO andmouse systems biological cellular will the using confirmation affected platelets from needsan independent family members. this 4, however shown figurein results western the analysis suggested by speculation is unstable G6Bprotein of the G6B native mutated product instead protein; this expressing an who likely family members, the of affected casein interrupted is cascade This article is This protectedAll rights byreserved. copyright. Accepted Article

9. Bouilloux F, Juban G, Cohet N, et al. EKLF restricts megakaryocytic differentiation at the benefit of of benefit the at differentiation megakaryocytic restricts EKLF al. et N, Cohet G, Juban F, Bouilloux 9. Crispino Weiss JD, MJ. Erythro-megakaryocytic transcription factorsassociated withhereditary 8. LC, Crispino Dore factor JD. Transcription networks cell erythroid andmegakaryocytein 7. VR, Tomer DeutschA. Megakaryocyte developmentproduction. and platelet Br HaematolJ 6. 5. Morris VK, Spraker HL, Howard SC, et al. Severe al. Spraker et HL, SC, VK, Howard Morris 5. BergerBrassSevere M,LF. thrombocytopeniairon in deficiencyanemia. HematolAm J 4. Levine RL, Hursting MJ,Drexler A, al.et Heparin-inducedthrombocytopenia in the emergency 3. BickRL. Disseminated intravascularcoagulation current concepts of etiology, pathophysiology, 2. Weinzierl ArberEP, DA. The differential diagnosis and bone marrow evaluation of new-onset 1. Genomics Unit, Genetics and Osama Alsmadi, : This article is This protectedAll rights byreserved. copyright. 2406. Email: 2406. Email: 4343, Fax:+ 2224 2999Ext. Phone:+965 Tel: Kuwait. 965 2249 Dasman15462, 1180, Accepted Article REFERENCES: erythrocytic differentiation. Blood 2008;112:576-584. 2008;112:576-584. Blood differentiation. erythrocytic Blood 2014;123:3080-3088. anemia. Blood 2011;118:231-239. development. 2006;134:453-466. 2010;27:413-419. Oncol Hematol Pediatr 1987;24:425-428. 1987;24:425-428. 2004;44:511-515. Med Emerg Ann department. 2003;17:149-176. Am North Clin Oncol Hematol treatment. and diagnosis, 2013;139:9-29. Pathol Clin J Am pancytopenia. [email protected] thrombocytopenia with iron deficiency anemia.

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degradation (E) lysosome inhibitor (chloroquine, 100 (chloroquine, inhibitor lysosome (E) degradation the of proteasome inhibition mild stability caused and inprotein increase amajor cause not did of truncated form the G6B in K562 untreated (C) cells. with 10Treating proteasome (MG132, inhibitor ( G6B. mutant and wild-type tagged FLAG of Expression 4. Figure cells). Flag transfected 6 day to relative **p<0.05 with compound; the incubation 6hours *p<0.05 to relative (n=3, 0 cells, day undifferentiated to as GAPDH relative afold-change and expressed were against normalized and results performed was qrtPCR points, time different at collected was RNA lineages. hematopoietic the induce G6B to failed molecules, inducing of absence In differentiation. erythrocyte- or platelet- of enhancement significant showed differentiation erythrocytes induce to Na-BU (B) or generation platelet induce to PMA with (A) vector treated ormonk mutant truncated-G6B wild type-G6B, with cells transfected Transiently Na-Bu. or PMA of presence the in cells K562 of differentiation lineage hematopoietic enhancing of G6B role functional 3.The Figure thisstate. have siblings change unaffected inheterozygous and Mother (p.C108*). mutation nonsense in a andresults exon, coding second the in located c.324C>A variant of AA genotype is 1 V showingII, homozygous andVII IV, participants affected of Chromatogram members. family all in showing genotypes and mutation the chromatograms Representative Figure 2. status. affection positive indicate Filled symbols females. represent symbol and males circle represent Square symbol status affection and siblings showing pedigree Family Figure1. Legends Figure This article is This protectedAll rights byreserved. copyright. protein. (B, D&F) transfection efficiency of the wild-type and the truncated form of G6B is measured at theat isG6B measured of form truncated wild-type andthe the of efficiency D&F) transfection (B, protein. Accepted of degradation the inshowing K562 G6Btransfected of forms tagged FLAG andA) truncated Wild-type Article

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A schematic diagram depicting G6B signaling [pathway in wild-type and truncated G6B. G6B. truncated and wild-type in [pathway signaling G6B depicting diagram Aschematic μ M) or (E) lysosome inhibitor (chloroquine, 100 (chloroquine, inhibitor lysosome or(E) M) μ M). Truncated form of G6B was stabilized by stabilizedby of was G6B form Truncated M).