The Research In Causative Genes Of Pediatric Henoch-Schonlein Purpura

Objective:The present study aims to explore the expression of C1GALT1 and Cosmc mRNA in peripheral blood mononuclear cell(PBMC)of Henoch-Schonlein purpura patients,as well as if Cosmc gene mutation exists in HSP patients and the association between Cosmc gene mutation and the susceptibility to HSP.In addition,the whole exome sequencing was performed between a boy with HSP and his father who suffered to HSP in his childhood.Meanwhile,the related causative genes were screened.Then the mutation of selected gene in 93 cases with HSP was detected.Methods:The peripheral blood samples and clinical data of 31 HSP patients were collected.Total RNA from PBMC was extracted using Trizol reagent.cDNA was synthesized using 1ug total RNA.Then real-time reverse transcriptase polymerase(RT-PCR)was performed to determine the expression of C1GALT1 and Cosmc mRNA in PBMC of HSP patients.Expanding the number of HSP patients to 84,genomic DNA was extracted using Trizol reagent.The whole exon region of Cosmc gene was amplified by touch-down PCR and then sequenced.The whole exome sequencing was firstly performed between a boy with HSP and his father who suffered to HSP in his childhood.Then,the related causative genes were screened.Finally,the mutation of selected gene in 93 cases with HSP was detected by using PCR.Results:HSP patients can be divided into non-HSPN group(27 cases)and HSPN4 cases)according to the renal involvement.The 2~-?Ct?Ct values of C1GALT1mRNA in control group?non-HSPN group and HSPN group were0.01716±0.00896?0.01392±0.00478?0.00999±0.00269 respectively and 2~-?Ct?Ct values of Cosmc mRNA of control group?non-HSPN group and HSPN group were 0.00319±0.00143?0.00266±0.00099?0.00241±0.00052.The expression level of C1GALT1 mRNA and Cosmc mRNA of non-HSPN group and HSPN group are lower than the control group,but there was no significant difference(p>0.05).Sequencing results showed that there were two sites of Cosmc gene mutation in HSP patients,which were c.393T>A and c.72 A>G;and the frequency distribution of every genotype and allele about the two mutant sites both had no significant difference in non-HSPN group?HSPN group and control group,so do in male samples and female samples.Whole genome sequencing result showed that MIF gene and MGAT5 gene may be related to the onset of HSP.The verification results in 93 children with HSP demonstrated that there were 3missense mutations of MIF gene(c.282-6C>G,c.338C>CT and c.295T>C).Then,3 synonymous mutations(c.142C>CT,c.348A>AG,c.267A>AG)and 2 missense mutations(c.1117A>AG,c.673C>G)of MGAT5 gene existed in 93 HSP patients.C.282-6C>G variant site was localized in the splicing region of MIF gene.The percentage of mutation site in HSP patients(37.6%)was close to that in the European East Asian(32.2%)and(28.6%).And the mutation site,which allele frequency is higher than 3%,was considered as a SNP site and may have no relation with the pathogenesis of HSP.In addition,two patients were found to have the c.338C>CT mutation site and one case had another mutation site c.295T>C.The mutation frequency of the two sites in HSP(2.15%,1.08%)is much higher than that of East Asian and European.Moreover,the three patients with severe clinical manifestation need adequate glucocorticoid or glucocorticoid combined with immunosuppressive,indicating that the gene mutation of MIF may be related to the incidence of HSP and its severity,glucocorticoid resistance,dosage and administration time.About the MGAT5 gene,synonymous mutation did not cause changes in amino acid and the structure and function of MGAT5protein,suggesting that there is no association with the pathogenesis of HSP.The c.1117A>AG and c.673C>G were found in one patient,respectively.Then the incidence of the two mutation sites in HSP(1.08%)is much higher than that of East Asian and European.The two mutations of HSP with severe symptoms need longer glucocorticoid administration time.It suggested that MGAT5 gene mutation may be associated with the severity of HSP and the administration time of glucocorticoid.Conclusions:The expression of C1GALT1 and Cosmc mRNA in PBMC of HSP patients may be more closely related with HSPN.Our data provide no evidence for a relevant association between the two mutant sites of Cosmc gene,c.393T>A and c.72 A>G,and the genetic susceptibility to HSP.The gene mutations of MIF and MGAT5 may be related to the incidence of HSP and its severity.Moreover,the gene mutation may also be associated with glucocorticoid resistance,dosage and administration time.