Study On GPⅠbα,GPⅢa,P2Y1and P2Y12Genetic Variants In Childhood Idiopathic Thrombocytopenic Purpura

Part One Study on GPⅠbα genetic variant in childhood idiopathicthrombocytopenic purpuraObjective:To research genetic variant of GPⅠbα （plateletglycoprotein Ib alpha， GPⅠbα)gene in childhood idiopathicthrombocytopenic purpura (ITP) in Guangxi Province, to investigate thecorrelation between childhood ITP pathogenesis and genetic variants,and to explore the pathogenesis of childhood ITP genetic mechanisms.Methods:(1) Collection in March2009to May2013in our hospitaloutpatient and the inpatient treatment group of76cases of childhood ITPcases. The control group included115healthy children.(2) Peripheralblood was collected to extract genomic DNA, and the exon2of GPⅠbαgene was analyzed by using the polymerase chain reaction (PCR)followed by direct sequencing.(3) genotype and allele distributions ofvariant were compared between cases and controls，Then clinical featureswere compared between C/T genotype and T/T genotype in the childhoodITP group. Results:(1) single-nucleotide polymorphism (SNP) loci rs6065（HPA-2）was identified in GPⅠbα gene.In the childhood ITP group C/Tgenotype （HPA-2ab）frequency was17.11%,while T allele （HPA-2b）frequency was8.55%.In the control group C/T genotype （HPA-2ab）frequency was3.48%,while T allele （HPA-2b）frequency was1.74%.(2)SNP loci rs6065（HPA-2） conformed to Hardy-Weinberg expectationsin the childhood ITP group and the control group. The genotype andallele frequency of rs6065（HPA-2）in the healthy population of Guangxiprovince was similar to studies based on Han Chinese, but significantlydifferent with Japanese, European and African (P<0.05).(3) rs6065（HPA-2）genotype and allele frequency distributions was significantlydifferent among childhood ITP patients and healthy controls (P<0.01).C/T genotype （HPA-2ab） was risk factor of the childhood ITP(P=0.001，OR:5.726,95%CI：1.791-18.312）,while T allele （HPA-2b）was risk factor of the childhood ITP (P=0.002，OR:5.284，95%CI：1.689-16.529）.(4)Clinical features between C/C （HPA-2aa）and C/Tgenotype （HPA-2ab） in childhood ITP group had no significantdifference.Conclusions:(1) GPⅠbα rs6065（HPA-2）has correlation withsusceptibility to childhood ITP in Guangxi Province. C/T genotype（HPA-2ab）and T allele （HPA-2b）are risk factors of the childhoodITP.(2) This is the first genotyping report of rs6065（HPA-2） in childrenof Guangxi Province. This finding help guiding platelet transfusion in thefuture. Part Two Study on GPⅢa genetic variants in childhood idiopathicthrombocytopenic purpuraObjective:To research genetic variants of GPⅢa (plateletglycoprotein Ⅲa) in childhood idiopathic thrombocytopenic purpura (ITP)in Guangxi Province, to investigate the correlation between childhoodITP pathogenesis and GPⅢa genetic variants, and to explore thepathogenesis of childhood ITP genetic mechanisms.Methods:(1) Collection in March2009to May2013in our hospitaloutpatient and the inpatient treatment group of70cases of childhood ITPcases. The control group included50healthy children.(2) Peripheralblood was collected to extract genomic DNA, and the exon1-15and theflanking intronic region around each exon of GPⅢa gene was analyzedby using the polymerase chain reaction (PCR) followed by directsequencing. The blood samples and genomic DNA of the control groupwere gained as above, and genotyping was carried out on mutationswhich were found in cases.(3) Genotype and allele distributions ofvariants were compared between cases and controls.Results:(1) A total of12variants were identified in GPⅢa gene,including one novel mutation D591H as well asrs5918,rs5917,rs15908,rs13306487,rs4642,rs4634,rs70940817,rs3760372,rs1969267,c.2015-61G>A and rs13306489were SNP loci,andc.2015-61G>A was new SNP loci.(2) All11SNP loci conformed to Hardy-Weinberg expectations in childhood ITP group.rs15908A>C,rs4642,rs4634,rs3760372,rs1969267,c.2015-61G>A andrs13306489conformed to Hardy-Weinberg expectations in bothchildhood ITP group and control group.(3) rs13306489genotype andallele frequency distributions was significantly different among childhoodITP patients and healthy controls (P<0.05). G/C genotype frequency andC allele frequency were decrease in childhood ITP group. genotype orallele frequency distributions of c.2015-61G>A,rs15908A>C,rs4642,rs4634,rs3760372and rs1969267were not significantly differentamong childhood ITP patients and healthy controls.Conclusions:(1) GPⅢa rs13306489has correlation withsusceptibility to childhood ITP in Guangxi Province. G/C genotypefrequency and C allele frequency were decrease in childhood ITP group.(2) D591H may be a novel mutation, and may be associated with theonset of the children with ITP. Rs70940817may be the commonpolymorphism loci of childhood ITP in Guangxi Province.(3)c.2015-61G>A is a novel SNP. c.2015-61G>A,rs15908A>C,rs4642,rs4634,rs3760372and rs1969267have no correlation withsusceptibility to childhood ITP. rs5918(HPA-1),rs5917(HPA-4) or rs13306487(HPA-6)variants may have no correlationwith susceptibility to childhood ITP. Part Three Study on P2Y1and P2Y12genetic variants in childhoodidiopathic thrombocytopenic purpuraObjective:To research genetic variants of P2Y1(Purinergic2receptor subtype Y1，P2Y1) and P2Y12(Purinergic2receptor subtype Y12，P2Y12)in childhood idiopathic thrombocytopenic purpura (ITP) inGuangxi Province, to investigate the correlation between childhood ITPpathogenesis and genetic variants, and to explore the pathogenesis ofchildhood ITP genetic mechanisms.Methods:(1) Collection in March2009to May2013in our hospitaloutpatient and the inpatient treatment group of71cases of childhood ITPcases, including33acute ITP patients and38chronic ITP patients.(2)Peripheral blood was collected to extract genomic DNA, and the exon1of P2Y1gene and exon2of P2Y12gene was analyzed by using thepolymerase chain reaction (PCR) followed by direct sequencing. Thecontrol group included50and69healthy children, whose blood samplesand genomic DNA were gained as above, and genotyping was carried outrespectively for P2Y1and P2Y12gene.(3) Genotype and alleledistributions of variants were compared between cases and controls.Results:(1) Four SNP loci were identified in P2Y1: rs1065776and rs199546439conformed to Hardy-Weinberg expectations in acute ITP group, while frequencies of rs118182811and rs701265conformed to Hardy-Weinberg expectations in all group.Genotype or allele distributions of rs118182811and rs701265had no significant difference among acute ITP patients, chronic ITP patients or healthy controls.(2) Two SNP loci rs6785930and rs6809699were found in P2Y12, and they conformed to Hardy-Weinberg expectations. genotype or allele frequency distributions of rs6785930and rs6809699werenot significantly different among acute ITP patients, chronic ITP patients or healthy controls.Conclusions:(1) P2Y1rs1065776and rs199546439may to beassociated with the onset of the children with ITP. P2Y1rs118182811orrs701265variants have no correlation with susceptibility to childhood ITP.(2) P2Y12rs6785930or rs6809699have no correlation withsusceptibility to childhood ITP.